Assays and methods using biomarkers

ABSTRACT

Methods and assays examining expression of one or more biomarkers in a mammalian tissue or cell sample are provided. According to the disclosed methods and assays, detection of the expression of one or more such biomarkers is predictive or indicative that the tissue or cell sample will be sensitive to apoptosis-inducing agents such as Apo2L/TRAIL and anti-DR5 agonist antibodies. Certain biomarkers which may be examined include fucosyltransferases, in particular fucosyltransferase 3 (FUT3) and/or fucosyltransferase 6 (FUT6), as well as sialyl Lewis A and/or X antigens. Kits and articles of manufacture are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/542,318, filed Oct. 3, 2006, which is a continuation ofPCT/US05/27480 filed Aug. 30, 2005 claiming priority under Section119(e) to provisional application No. 60/599,425 filed Aug. 6, 2004, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention described herein relates to methods and assays to detectbiomarkers predictive of sensitivity of mammalian cells to Apo2L/TRAILand/or death receptor agonist antibodies.

BACKGROUND OF THE INVENTION

Various ligands and receptors belonging to the tumor necrosis factor(TNF) superfamily have been identified in the art. Included among suchligands are tumor necrosis factor-alpha (“TNF-alpha”), tumor necrosisfactor-beta (“TNF-beta” or “lymphotoxin-alpha”), lymphotoxin-beta(“LT-beta”), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BBligand, LIGHT, Apo-1 ligand (also referred to as Fas ligand or CD95ligand), Apo-2 ligand (also referred to as Apo2L or TRAIL), Apo-3 ligand(also referred to as TWEAK), APRIL, OPG ligand (also referred to as RANKligand, ODF, or TRANCE), and TALL-1 (also referred to as BlyS, BAFF orTHANK) (See, e.g., Ashkenazi, Nature Review, 2:420-430 (2002); Ashkenaziand Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit, Curr.Opin. Cell Biol., 11:255-260 (2000); Golstein, Curr. Biol., 7:750-753(1997) Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411;Locksley et al., Cell, 104:487-501 (2001); Gruss and Dower, Blood,85:3378-3404 (1995); Schmid et al., Proc. Natl. Acad. Sci., 83:1881(1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987); Pitti et al.,J. Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity,3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993); Armitage etal. Nature, 357:80-82 (1992); WO 97/01633 published Jan. 16, 1997; WO97/25428 published Jul. 17, 1997; Marsters et al., Curr. Biol.,8:525-528 (1998); Chicheportiche et al., Biol. Chem., 272:32401-32410(1997); Hahne et al., J. Exp. Med., 188:1185-1190 (1998); WO98/28426published Jul. 2, 1998; WO98/46751 published Oct. 22, 1998; WO/98/18921published May 7, 1998; Moore et al., Science, 285:260-263 (1999); Shu etal., J. Leukocyte Biol., 65:680 (1999); Schneider et al., J. Exp. Med.,189:1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem.,274:15978-15981 (1999)).

Induction of various cellular responses mediated by such TNF familyligands is typically initiated by their binding to specific cellreceptors. Some, but not all, TNF family ligands bind to, and inducevarious biological activity through, cell surface “death receptors” toactivate caspases, or enzymes that carry out the cell death or apoptosispathway (Salvesen et al., Cell, 91:443-446 (1997). Included among themembers of the TNF receptor superfamily identified to date are TNFR1,TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also referred toas Apo-1 or CD95), DR4 (also referred to as TRAIL-R1), DR5 (alsoreferred to as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG),RANK and Apo-(also referred to as DR3 or TRAMP) (see, e.g., Ashkenazi,Nature Reviews, 2:420-430 (2002); Ashkenazi and Dixit, Science,281:1305-1308 (1998); Ashkenazi and Dixit, Curr. Opin. Cell Biol.,11:255-260 (2000); Golstein, Curr. Biol., 7:750-753 (1997) Wallach,Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley etal., Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404(1995); Hohman et al., J. Biol. Chem., 264:14927-14934 (1989); Brockhauset al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563,published Mar. 20, 1991; Loetscher et al., Cell, 61:351 (1990); Schallet al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023(1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991);Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991); Stamenkovic etal., EMBO J., 8:1403-1410 (1989); Mallett et al., EMBO J., 9:1063-1068(1990); Anderson et al., Nature, 390:175-179 (1997); Chicheportiche etal., J. Biol. Chem., 272:32401-32410 (1997); Pan et al., Science,276:111-113 (1997); Pan et al., Science, 277:815-818 (1997); Sheridan etal., Science, 277:818-821 (1997); Degli-Esposti et al., J. Exp. Med.,186:1165-1170 (1997); Marsters et al., Curr. Biol., 7:1003-1006 (1997);Tsuda et al., BBRC, 234:137-142 (1997); Nocentini et al., Proc. Natl.Acad. Sci., 94:6216-6221 (1997); vonBulow et al., Science, 278:138-141(1997)).

Most of these TNF receptor family members share the typical structure ofcell surface receptors including extracellular, transmembrane andintracellular regions, while others are found naturally as solubleproteins lacking a transmembrane and intracellular domain. Theextracellular portion of typical TNFRs contains a repetitive amino acidsequence pattern of multiple cysteine-rich domains (CRDs), starting fromthe NH₂-terminus.

The ligand referred to as Apo-2L or TRAIL was identified several yearsago as a member of the TNF family of cytokines. (see, e.g., Wiley etal., Immunity, 3:673-682 (1995); Pitti et al., J. Biol. Chem.,271:12697-12690 (1996); WO 97/01633; WO 97/25428; U.S. Pat. No.5,763,223 issued Jun. 9, 1998; U.S. Pat. No. 6,284,236 issued Sep. 4,2001). The full-length native sequence human Apo2L/TRAIL polypeptide isa 281 amino acid long, Type II transmembrane protein. Some cells canproduce a natural soluble form of the polypeptide, through enzymaticcleavage of the polypeptide's extracellular region (Mariani et al., J.Cell. Biol., 137:221-229 (1997)). Crystallographic studies of solubleforms of Apo2L/TRAIL reveal a homotrimeric structure similar to thestructures of TNF and other related proteins (Hymowitz et al., Molec.Cell, 4:563-571 (1999); Cha et al., Immunity, 11:253-261 (1999);Mongkolsapaya et al., Nature Structural Biology, 6:1048 (1999); Hymowitzet al., Biochemistry, 39:633-644 (2000)). Apo2L/TRAIL, unlike other TNFfamily members however, was found to have a unique structural feature inthat three cysteine residues (at position 230 of each subunit in thehomotrimer) together coordinate a zinc atom, and that the zinc bindingis important for trimer stability and biological activity. (Hymowitz etal., supra; Bodmer et al., J. Biol. Chem., 275:20632-20637 (2000)).

It has been reported in the literature that Apo2L/TRAIL may play a rolein immune system modulation, including autoimmune diseases such asrheumatoid arthritis [see, e.g., Thomas et al., J. Immunol.,161:2195-2200 (1998); Johnsen et al., Cytokine, 11:664-672 (1999);Griffith et al., J. Exp. Med., 189:1343-1353 (1999); Song et al., J.Exp. Med., 191:1095-1103 (2000)].

Soluble forms of Apo2L/TRAIL have also been reported to induce apoptosisin a variety of cancer cells, including colon, lung, breast, prostate,bladder, kidney, ovarian and brain tumors, as well as melanoma,leukemia, and multiple myeloma (see, e.g., Wiley et al., supra; Pitti etal., supra; U.S. Pat. No. 6,030,945 issued Feb. 29, 2000; U.S. Pat. No.6,746,668 issued Jun. 8, 2004; Rieger et al., FEBS Letters, 427:124-128(1998); Ashkenazi et al., J. Clin. Invest., 104:155-162 (1999); Walczaket al., Nature Med., 5:157-163 (1999); Keane et al., Cancer Research,59:734-741 (1999); Mizutani et al., Clin. Cancer Res., 5:2605-2612(1999); Gazitt, Leukemia, 13:1817-1824 (1999); Yu et al., Cancer Res.,60:2384-2389 (2000); Chinnaiyan et al., Proc. Natl. Acad. Sci.,97:1754-1759 (2000)). In vivo studies in murine tumor models furthersuggest that Apo2L/TRAIL, alone or in combination with chemotherapy orradiation therapy, can exert substantial anti-tumor effects (see, e.g.,Ashkenazi et al., supra; Walzcak et al., supra; Gliniak et al., CancerRes., 59:6153-6158 (1999); Chinnaiyan et al., supra; Roth et al.,Biochem. Biophys. Res. Comm., 265:1999 (1999); PCT ApplicationUS/00/15512; PCT Application US/01/23691). In contrast to many types ofcancer cells, most normal human cell types appear to be resistant toapoptosis induction by certain recombinant forms of Apo2L/TRAIL(Ashkenazi et al., supra; Walzcak et al., supra). Jo et al. has reportedthat a polyhistidine-tagged soluble form of Apo2L/TRAIL inducedapoptosis in vitro in normal isolated human, but not non-human,hepatocytes (Jo et al., Nature Med., 6:564-567 (2000); see also, Nagata,Nature Med., 6:502-503 (2000)). It is believed that certain recombinantApo2L/TRAIL preparations may vary in terms of biochemical properties andbiological activities on diseased versus normal cells, depending, forexample, on the presence or absence of a tag molecule, zinc content, and% trimer content (See, Lawrence et al., Nature Med., Letter to theEditor, 7:383-385 (2001); Qin et al., Nature Med., Letter to the Editor,7:385-386 (2001)).

Apo2L/TRAIL has been found to bind at least five different receptors. Atleast two of the receptors which bind Apo2L/TRAIL contain a functional,cytoplasmic death domain. One such receptor has been referred to as“DR4” (and alternatively as TR4 or TRAIL-R1) (Pan et al., Science,276:111-113 (1997); see also WO98/32856 published Jul. 30, 1998;WO99/37684 published Jul. 29, 1999; WO 00/73349 published Dec. 7, 2000;U.S. Pat. No. 6,433,147 issued Aug. 13, 2002; U.S. Pat. No. 6,461,823issued Oct. 8, 2002, and U.S. Pat. No. 6,342,383 issued Jan. 29, 2002).

Another such receptor for Apo2L/TRAIL has been referred to as DR5 (ithas also been alternatively referred to as Apo-2; TRAIL-R or TRAIL-R2,TR6, Tango-63, hAPO8, TRICK2 or KILLER) (see, e.g., Sheridan et al.,Science, 277:818-821 (1997), Pan et al., Science, 277:815-818 (1997),WO98/51793 published Nov. 19, 1998; WO98/41629 published Sep. 24, 1998;Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J.,16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997);WO98/35986 published Aug. 20, 1998; EP870,827 published Oct. 14, 1998;WO98/46643 published Oct. 22, 1998; WO99/02653 published Jan. 21, 1999;WO99/09165 published Feb. 25, 1999; WO99/11791 published Mar. 11, 1999;US 2002/0072091 published Aug. 13, 2002; US 2002/0098550 published Dec.7, 2001; U.S. Pat. No. 6,313,269 issued Dec. 6, 2001; US 2001/0010924published Aug. 2, 2001; US 2003/01255540 published Jul. 3, 2003; US2002/0160446 published Oct. 31, 2002, US 2002/0048785 published Apr. 25,2002; U.S. Pat. No. 6,342,369 issued February, 2002; U.S. Pat. No.6,569,642 issued May 27, 2003, U.S. Pat. No. 6,072,047 issued Jun. 6,2000, U.S. Pat. No. 6,642,358 issued Nov. 4, 2003; IS 6,743,625 issuedJun. 1, 2004). Like DR4, DR5 is reported to contain a cytoplasmic deathdomain and be capable of signaling apoptosis upon ligand binding (orupon binding a molecule, such as an agonist antibody, which mimics theactivity of the ligand). The crystal structure of the complex formedbetween Apo-2L/TRAIL and DR5 is described in Hymowitz et al., MolecularCell, 4:563-571 (1999).

Upon ligand binding, both DR4 and DR5 can trigger apoptosisindependently by recruiting and activating the apoptosis initiator,caspase-8, through the death-domain-containing adaptor molecule referredto as FADD/Mort1 [Kischkel et al., Immunity, 12:611-620 (2000); Spricket al., Immunity, 12:599-609 (2000); Bodmer et al., Nature Cell Biol.,2:241-243 (2000)].

Apo2L/TRAIL has been reported to also bind those receptors referred toas DcR1, DcR2 and OPG, which believed to function as inhibitors, ratherthan transducers of signaling (see., e.g., DCR1 (also referred to asTRID, LIT or TRAIL-R3) [Pan et al., Science, 276:111-113 (1997);Sheridan et al., Science, 277:818-821 (1997); McFarlane et al., J. Biol.Chem., 272:25417-25420 (1997); Schneider et al., FEBS Letters,416:329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186:1165-1170(1997); and Mongkolsapaya et al., J. Immunol., 160:3-6 (1998); DCR2(also called TRUNDD or TRAIL-R4) [Marsters et al., Curr. Biol.,7:1003-1006 (1997); Pan et al., FEBS Letters, 424:41-45 (1998);Degli-Esposti et al., Immunity, 7:813-820 (1997)], and OPG [Simonet etal., supra]. In contrast to DR4 and DR5, the DcR1 and DcR2 receptors donot signal apoptosis.

Certain antibodies which bind to the DR4 and/or DR5 receptors have beenreported in the literature. For example, anti-DR4 antibodies directed tothe DR4 receptor and having agonistic or apoptotic activity in certainmammalian cells are described in, e.g., WO 99/37684 published Jul. 29,1999; WO 00/73349 published Jul. 12, 2000; WO 03/066661 published Aug.14, 2003. See, also, e.g., Griffith et al., J. Immunol., 162:2597-2605(1999); Chuntharapai et al., J. Immunol., 166:4891-4898 (2001); WO02/097033 published Dec. 2, 2002; WO 03/042367 published May 22, 2003;WO 03/038043 published May 8, 2003; WO 03/037913 published May 8, 2003.Certain anti-DR5 antibodies have likewise been described, see, e.g., WO98/51793 published Nov. 8, 1998; Griffith et al., J. Immunol.,162:2597-2605 (1999); Ichikawa et al., Nature Med., 7:954-960 (2001);Hylander et al., “An Antibody to DR5 (TRAIL-Receptor 2) Suppresses theGrowth of Patient Derived Gastrointestinal Tumors Grown in SCID mice”,Abstract, 2d International Congress on Monoclonal Antibodies in Cancers,Aug. 29-Sep. 1, 2002, Banff, Alberta, Canada; WO 03/038043 published May8, 2003; WO 03/037913 published May 8, 2003. In addition, certainantibodies having cross-reactivity to both DR4 and DR5 receptors havebeen described (see, e.g., U.S. Pat. No. 6,252,050 issued Jun. 26,2001).

Neoplastic transformation of some mammalian cells has in certaininstances, been associated with characteristic changes in the expressionof sialyl Lewis A and sialyl Lewis X antigens. Relatively high amountsof sialyl Lewis A/X are present, for example, in some humanadenocarcinomas of the colon, pancreas and stomach, and assays usingantibodies directed to the carbohydrate structures on these antigenshave been employed as a means to detect pancreatic and gastrointestinalcancers. (see, e.g., Ugorski et al., Acta Biochimica Polonica,49:2:303-311 (2002). The level of expression of these carbohydrate tumormarkers has also been correlated with clinical outcome, patient survivaltimes and an indicator of metastatic disease.

Both sialyl Lewis A and sialyl Lewis X have been shown to bind to afamily of carbohydrate-binding proteins involved in the extravasation ofcells from the bloodstream, called the selectins. Some reports suggestthat sialyl Lewis A and X are ligands for E-selectin, and may beresponsible for the adhesion of human cancer cells to endothelium.Sialylated Lewis structures present on the surface of cancer cells arecarried by the carbohydrate chains of glycoproteins and glycolipids andbind E-selectin present on endothelial cells. Selectins and theircarbohydrate ligands may accordingly play an important role in theselective homing of tumor cells during metastasis.

The biosynthesis of sialyl Lewis A and X is believed to be dependentupon the final addition of fucose from guanosine diphosphate-fucose(GDP-Fuc) in alpha (1,3) and alpha (1,4) linkage to sialylatedprecursors by cell type-specific and developmental stage-specificenzymes, a step catalyzed by alpha-1,3/1,4-fucosyltransferases (alpha1,3/1,4 Fuc-T, FUT).

Several human fucosyltransferase genes have been cloned andcharacterized to date. Expression of these genes (FUT 3-7) and theirenzyme products (Fuc-TIII-VII) appears to be tissue specific. Theenzymes encoded by the five genes are named FUTIII, FUTIV, FUTV, FUTVIand FUTVII. The three genes encoding FUTIII, FUTV and FUTVI arelocalized at close physical positions on chromosome 19p13.3. Biochemicaland molecular cloning studies suggest that lineage-specific expressionof the sialyl Lewis A/X moiety is determined by lineage-specificexpression of alpha-1,3-fucosyltransferase genes, whose enzyme productsoperate on constitutively expressed oligosaccharide precursors to yieldsurface-localized sialyl Lewis A/X determinants. The humanfucosyltransferases responsible for activity in epithelial tissues areFUT3 and FUT6. FUT3 [also called the Lewis alpha(1,3/1,4)fucosyltransferase gene] and FUT6 [the plasma alpha(1,3)fucosyltransferase gene] transcripts are present in both normal andtransformed tissues. Fucosyltransferase transcripts are also prevalentin numerous adenocarcinoma cell lines, with notably high expression ofFUT3 and 6 in colon carcinoma. (see, e.g, Ugorski et al., ActaBiochimica Polonica, 49:303-311 (2002); Nakamori et al., Dis. ColonRectum., 40:420-431 (1997); Takada et al., Cancer Res., 53:354-361(1993); Ichikawa et al., J. Surg. Oncol., 75:98-102 (2000)); Nakagoe etal., J Exp Clin Cancer Res., 2002 March; 21 (1):107-13; Matsumoto etal., Br J. Cancer. 2002 Jan. 21; 86 (2):161-7; Ito et al., J.Gastroenterol. 2001 December; 36 (12):823-9; Nakagoe et al., CancerDetect Prev. 2001; 25 (3):299-308; Kumamoto et al., Cancer Res. 2001Jun. 1; 61 (11):4620-7; Murata et al., Dis Colon Rectum. 2001 April; 44(4):A2-A4; Nakagoe et al., J Exp Clin Cancer Res. 2001 March; 20(1):85-90; Nakagoe et al., J. Gastroenterol. 2001 March; 36 (3):166-72;Nakagoe et al., Tumour Biol. 2001 March-April; 22 (2):115-22; Nakagoe etal., Can J. Gastroenterol. 2000 October; 14 (9):753-60; Izawa et al.,Cancer Res. 2000 Mar. 1; 60 (5):1410-6; Tanaka et al.,Hepatogastroenterology. 1999 March-April; 46 (26):875-82; Matsushita etal., Cancer Lett. 1998 Nov. 27; 133 (2):151-60; Sato et al., AnticancerRes. 1997 September-October; 17 (5A):3505-11; Yamada et al., Br J.Cancer. 1997; 76 (5):582-7; Nakamori et al., Dis Colon Rectum. 1997April; 40 (4):420-31; Srinivas et al., Scand J. Immunol. 1996 September;44 (3):197-203; Matsushita et al., Lab Invest. 1990 December; 63(6):780-91; Ashizawa et al., J Exp Clin Cancer Res. 2003 March; 22(1):91-8; Nakagoe et al., J Exp Clin Cancer Res. 2002 September; 21(3):363-9; Nakagoe et al., Anticancer Res. 2002 January-February; 22(1A):451-8; Nakagoe et al., J Clin Gastroenterol. 2002 April; 34(4):408-15; Nakagoe et al., Cancer Lett. 2002 Jan. 25; 175 (2):213-21;Tatsumi et al., Clin Exp Metastasis. 1998 November; 16 (8):743-50; Ikedaet al., J Surg Oncol. 1996 July; 62 (3):171-6; Ikeda et al., Eur J SurgOncol. 1995 April; 21 (2):168-75; Togayachi et al., Int J. Cancer. 1999Sep. 24; 83 (1):70-9; Satoh et al., Clin Cancer Res. 1997 April; 3(4):495-9; Satoh et al., Respiration. 1998; 65 (4):295-8; Satoh et al.,Anticancer Res. 1998 July-August; 18 (4B):2865-8; Fukuoka et al., LungCancer. 1998 May; 20 (2):109-16; Fujiwara et al., Anticancer Res. 1998March-April; 18 (2A):1043-6; Ogawa et al., Int J. Cancer. 1997 Apr. 22;74 (2):189-92; Ogawa et al., J Thorac Cardiovasc Surg. 1994 August; 108(2):329-36; Asao et al., Cancer. 1989 Dec. 15; 64 (12):2541-5; Narita etal., Breast Cancer. 1996 Mar. 29; 3 (1):19-23; Yamaguchi et al.,Oncology. 1998 July-August; 55 (4):357-62; Sikut et al., Int J. Cancer.1996 May 29; 66 (5):617-23; Saito et al., Anticancer Res. 2003July-August; 23 (4):3441-6; Fujii et al., Urol Int. 2000; 64 (3):129-33;Idikio et al., Glycoconj J. 1997 November; 14 (7):875-7; Inoue et al.,Obstet. Gynecol. 1992 March; 79 (3):434-40; Yamashita et al., Eur JCancer. 2000 January; 36 (1):113-20; Hamanaka et al., Pancreas. 1996August; 13 (2):160-5; Ho et al., Cancer Res. 1995 Aug. 15; 55(16):3659-63.

SUMMARY OF THE INVENTION

The invention disclosed herein provides methods and assays examiningexpression of one or more biomarkers in a mammalian tissue or cellsample, wherein the expression of one or more such biomarkers ispredictive of whether the tissue or cell sample will be sensitive toapoptosis-inducing agents such as Apo2L/TRAIL and anti-DR5 agonistantibodies. In various embodiments of the invention, the methods andassays examine expression of biomarkers such as certainfucosyltransferases, in particular fucosyltransferase 3 (FUT3) and/orfucosyltransferase 6 (FUT6), as well as sialyl Lewis A and/or Xantigens.

As discussed above, most normal human cell types appear to be resistantto apoptosis induction by certain recombinant forms of Apo2L/TRAIL(Ashkenazi et al., supra; Walzcak et al., supra). It has also beenobserved that some populations of diseased human cell types (such ascertain populations of cancer cells) are resistant to apoptosisinduction by certain recombinant forms of Apo2L/TRAIL (Ashkenazi et al.,J. Clin. Invest., 1999, supra; Walczak et al., Nature Med., 1999,supra). Consequently, by examining a mammalian tissue or cell sample forexpression of certain biomarkers by way of an assay, one canconveniently and efficiently obtain information useful in assessingappropriate or effective therapies for treating patients. For example,information obtained from an assay to detect FUT3 or FUT6 expression ina mammalian tissue or cell sample can provide physicians with usefuldata that can be used to determine an optimal therapeutic regimen (usingApo2L/TRAIL or death receptor agonist antibodies) for patients sufferingfrom a disorder such as cancer.

The invention provides methods for predicting the sensitivity of amammalian tissue or cells sample (such as a cancer cell) to Apo2L/TRAILor a death receptor agonist antibody. In certain embodiments, themethods comprise obtaining a mammalian tissue or cell sample andexamining the tissue or cell for expression of fucosyltransferase 3 orfucosyltransferase 6. The methods may also comprise examining the tissueor cell for expression of another biomarker such as sialyl Lewis Aand/or X antigen(s). The methods may be conducted in a variety of assayformats, including assays detecting mRNA expression, enzymatic assaysdetecting presence of enzymatic activity, immunohistochemistry assays,and others discussed herein. Determination of expression of suchbiomarkers in said tissues or cells will be predictive that such tissuesor cells will be sensitive to the apoptosis-inducing activity ofApo2/TRAIL and/or death receptor antibody. In optional embodiments, thetissues or cells may also be examined for expression of DR4, DR5, DcR1or DcR2 receptors.

Further methods of the invention include methods of inducing apoptosisin a mammalian tissue or cell sample, comprising steps of obtaining amammalian tissue or cell sample, examining the tissue or cell forexpression of one or more biomarkers, such as fucosyltransferase 3,fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), and upondetermining said tissue or cell sample expresses said one or morebiomarkers, exposing said tissue or cell sample to an effective amountof Apo2L/TRAIL or death receptor agonist antibody. The steps in themethods for examining expression of one or more biomarkers may beconducted in a variety of assay formats, including assays detecting mRNAexpression, enzymatic assays detecting presence of enzymatic activity,and immunohistochemistry assays. In optional embodiments, the methodsalso comprise examining the tissue or cell sample for expression of DR4,DR5, DcR1, or DcR2 receptors. Optionally, the tissue or cell samplecomprises cancer tissue or cells.

Still further methods of the invention include methods of treating adisorder in a mammal, such as an immune related disorder or cancer,comprising steps of obtaining tissue or a cell sample from the mammal,examining the tissue or cells for expression of one or more biomarkers,such as fucosyltransferase 3, fucosyltransferase 6, sialyl Lewis Aand/or X antigen(s), and upon determining said tissue or cell sampleexpresses said one or more biomarkers, administering an effective amountof Apo2L/TRAIL or death receptor agonist antibody to said mammal. Thesteps in the methods for examining expression of one or more biomarkersmay be conducted in a variety of assay formats, including assaysdetecting mRNA expression, enzymatic assays detecting presence ofenzymatic activity, and immunohistochemistry assays. In optionalembodiments, the methods also comprise examining the tissue or cellsample for expression of DR4, DR5, DcR1, or DcR2 receptors. Optionally,the methods comprise treating cancer in a mammal. Optionally, themethods comprise, in addition to administering an effective amount ofApo2L/TRAIL and/or death receptor agonist antibody, administeringchemotherapeutic agent(s) or radiation therapy to said mammal.

Further embodiments are more particularly disclosed by the following

-   1. A method for predicting the sensitivity of a mammalian tissue or    cells sample to Apo2L/TRAIL, comprising the steps of:-   obtaining a mammalian tissue or cell sample;-   examining the tissue or cell sample to detect expression of one or    more biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), wherein    expression of said one or more biomarkers is predictive that said    tissue or cell sample is sensitive to apoptosis-inducing activity of    Apo2L/TRAIL.-   2. The method of claim 1 wherein said expression of one or more    biomarkers is examined by detecting mRNA expression of    fucosyltransferase 3 or fucosyltransferase 6.-   3. The method of claim 1 wherein said expression of one or more    biomarkers is examined by immunohistochemistry to detect expression    of sialyl Lewis A and/or X antigen(s).-   4. The method of claim 1 further comprising the step of examining    expression of DR4, DR5, DcR1, or DcR2 receptors in said tissue or    cell sample.-   5. The method of claim 1 wherein tissue or cell sample comprises    cancer tissue or cells.-   6. The method of claim 5 wherein said cancer cells are colon,    colorectal, gastrointestinal, or pancreatic cancer cells or tissue.-   7. A method for inducing apoptosis in a mammalian tissue or cell    sample, comprising the steps of:-   obtaining a mammalian tissue or cell sample;-   examining the tissue or cell sample to detect expression of one or    more biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), and    subsequent to detecting expression of said one or more biomarkers,    exposing said tissue or cell sample to an effective amount of    Apo2L/TRAIL.-   8. The method of claim 7 wherein said expression of one or more    biomarkers is examined by testing for mRNA expression of    fucosyltransferase 3 or fucosyltransferase 6.-   9. The method of claim 7 wherein said expression of one or more    biomarkers is examined by immunohistochemistry to detect expression    of sialyl Lewis A and/or X antigen(s).-   10. The method of claim 7 further comprising the step of examining    expression of DR4, DR5, DcR1 or DcR2 receptors in said tissue or    cell sample.-   11. The method of claim 7 wherein said tissue or cell sample    comprises cancer tissue or cells.-   12. The method of claim 11 wherein said cancer cells are colon,    colorectal, gastrointestinal, or pancreatic cancer cells or tissue.-   13. The method of claim 7 wherein said cells are exposed to an    effective amount of Apo2L/TRAIL polypeptide comprising amino acids    114-281 of FIG. 1.-   14. A method of treating a disorder in a mammal, such as an immune    related disorder or cancer, comprising the steps of:-   obtaining a tissue or cell sample from said mammal;-   examining the tissue or cell sample to detect expression of one or    more biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), and    subsequent to detecting expression of said one or more biomarkers,    administering to said mammal an effective amount of Apo2L/TRAIL.-   15. The method of claim 14 wherein said expression of one or more    biomarkers is examined by detecting mRNA expression of    fucosyltransferase 3 or fucosyltransferase 6.-   16. The method of claim 14 wherein said expression of one or more    biomarkers is examined by immunohistochemistry to detect expression    of sialyl Lewis A and/or X antigen(s).-   17. The method of claim 14 further comprising the step of examining    expression of DR4, DR5, DcR1 or DcR2 receptors in said tissue or    cell.-   18. The method of claim 14 wherein tissue or cell sample comprises    cancer tissue or cells.-   19. The method of claim 18 wherein said cancer cells or tissue    comprises colon, colorectal, gastrointestinal, or pancreatic cancer    cells or tissue.-   20. The method of claim 14 wherein an effective amount of    Apo2L/TRAIL polypeptide comprising amino acids 114-281 of FIG. 1 is    administered to said mammal.-   21. The method of claim 14 wherein a chemotherapeutic agent(s) or    radiation therapy is also administered to said mammal.-   22. The method of claim 14 wherein a cytokine, cytotoxic agent or    growth inhibitory agent is also administered to said mammal.-   23. The method of claim 7 wherein said Apo2L/TRAIL polypeptide is    linked to a polyethylene glycol molecule.-   24. The method of claim 14 wherein said Apo2L/TRAIL polypeptide is    linked to a polyethylene glycol molecule.-   25. The method of claim 6 wherein said cancer cells are colon or    colorectal cancer cells.-   26. The method of claim 1 wherein said Apo2L/TRAIL is a polypeptide    comprising amino acids 41-281 of FIG. 1 (SEQ ID NO:1) or a    biologically active fragment thereof.-   27. The method of claim 26 wherein said Apo2L/TRAIL is a polypeptide    comprising amino acids 114-281 of FIG. 1 (SEQ ID NO:1).-   28. The method of claim 12 wherein said cancer cells are colon or    colorectal cancer cells.-   29. The method of claim 20 wherein said Apo2L/TRAIL polypeptide    consists of amino acids 114-281 of FIG. 1 (SEQ ID NO:1).-   30. A method for predicting the sensitivity of a mammalian colon or    colorectal cancer cells to Apo2L/TRAIL, comprising the steps of:-   obtaining mammalian colon or colorectal cancer cells;-   examining the cancer cells to detect expression of one or more    biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), wherein    expression of said one or more biomarkers is predictive that said    cancer cells are sensitive to apoptosis-inducing activity of    Apo2L/TRAIL.-   31. A method for inducing apoptosis in mammalian colon or colorectal    cancer cells, comprising the steps of:-   obtaining mammalian colon or colorectal cancer cells;-   examining the cancer cells to detect expression of one or more    biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), and-   subsequent to detecting expression of said one or more biomarkers,    exposing said cancer cells to an effective amount of Apo2L/TRAIL    polypeptide.-   32. The method of claim 31 wherein said Apo2L/TRAIL polypeptide    comprises amino acids 41-281 of FIG. 1 (SEQ ID NO:1) or a fragment    thereof which has apoptotic activity.-   33. The method of claim 32 wherein said Apo2L/TRAIL polypeptide is    linked to a polyethylene glycol molecule.-   34. The method of claim 32 wherein said Apo2L/TRAIL polypeptide    comprises amino acids 114-281 of FIG. 1 (SEQ ID NO:1).-   35. A method of treating colon or colorectal cancer in a mammal,    comprising the steps of:-   obtaining a colon or colorectal cancer cell sample from said mammal;-   examining the cancer cell sample to detect expression of one or more    biomarkers selected from the group of fucosyltransferase 3,    fucosyltransferase 6, sialyl Lewis A and/or X antigen(s), and    subsequent to detecting expression of said one or more biomarkers,    administering to said mammal an effective amount of Apo2L/TRAIL.-   36. The method of claim 35 wherein said Apo2L/TRAIL polypeptide    comprises amino acids 41-281 of FIG. 1 (SEQ ID NO:1) or a fragment    thereof which has apoptotic activity.-   37. The method of claim 36 wherein said Apo2L/TRAIL polypeptide is    linked to a polyethylene glycol molecule.-   38. The method of claim 36 wherein said Apo2L/TRAIL polypeptide    comprises amino acids 114-281 of FIG. 1 (SEQ ID NO:1).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ IDNO:2) and its derived amino acid sequence (SEQ ID NO:1). The “N” atnucleotide position 447 is used to indicate the nucleotide base may be a“T” or “G”.

FIGS. 2A and 2B show the nucleotide sequence of a cDNA (SEQ ID NO:4) forfull length human DR4 and its derived amino acid sequence (SEQ ID NO:3).The respective nucleotide and amino acid sequences for human DR4 arealso reported in Pan et al., Science, 276:111 (1997).

FIG. 3A shows the 411 amino acid sequence (SEQ ID NO:5) of human DR5 aspublished in WO 98/51793 on Nov. 19, 1998. A transcriptional splicevariant of human DR5 is known in the art. This DR5 splice variantencodes the 440 amino acid sequence (SEQ ID NO:6) of human DR5 shown inFIGS. 3B and 3C as published in WO 98/35986 on Aug. 20, 1998.

FIG. 3D shows the nucleotide sequences of cDNA (SEQ ID NO:7) for fulllength human DcR1 and its derived amino acid sequence (SEQ ID NO:8). Therespective nucleotide and amino acid sequences for human DcR1 (andparticular domains thereof) are also shown and described in WO 98/58062.

FIG. 3E shows the nucleotide sequences of cDNA (SEQ ID NO:9) for fulllength human DcR2 and its derived amino acid sequence (SEQ ID NO:10).The respective nucleotide and amino acid sequences for human DcR2 (andparticular domains thereof) are shown in WO 99/10484.

FIG. 4 shows the nucleotide sequence of a cDNA (SEQ ID NO:11) for fulllength human (1,3/1,4) fucosyltransferase (FUT3) and its derived aminoacid sequence (SEQ ID NO:12). These sequences correspond to GenBankAccession Number HSU27328 and are described for example inKukowska-Latallo et al., Genes Dev. 1990 August; 4 (8):1288-303.

FIG. 5 shows the nucleotide sequence of a cDNA (SEQ ID NO:13) for fulllength human alpha (1,3) fucosyltransferase (FUT6) and its derived aminoacid sequence (SEQ ID NO:14). These sequences correspond to GenBankAccession Number HSU27333 and are described for example in Koszdin andBowen, Biochem Biophys Res Commun. 1992 Aug. 31; 187 (1):152-7.

FIG. 6 provides a summary chart of the data obtained in analyzing 28colon or colorectal cancer cell lines for sensitivity or resistance toapoptotic activity of Apo2L (+0.5% fetal bovine serum “FBS” or 10% FBS)or DR5 monoclonal antibody “mab”, cross-linked “XL” or not crosslinked,+0.5% fetal bovine serum “FBS” or 10% FBS) and expression of FUT 3, FUT6, Sialyl lewis A and Sialyl lewis X.

FIG. 7 provides a comparison of sensitivity of various colon orcolorectal cancer cell lines to DR5 antibody and the expression of FUT3, as measured by quantitative PCR.

FIG. 8 provides a comparison of various colon or colorectal cancer celllines for sensitivity or resistance to DR5 antibody (plus cross-linker)and expression of sialyl lewis X or A, as determined by FACS.

FIG. 9A shows a Spearman Rank Correlation test analyzing sensitivity orresistance of various colon or colorectal cancer cell lines andcorrelation to expression of FUT3.

FIG. 9B shows the results of a Fisher's Exact test analyzing sensitivity(“sens”) or resistance (“res”) of the various colon or colorectal cancercell lines and the statistical significance between FUT 3 and sialyllewis A/X expression and sensitivity of the respective cell lines to DR5antibody apoptotic activity.

FIG. 10 provides a comparison of various colon or colorectal cancer celllines for expression of DcR1 or DcR2 receptors (as determined byquantitative PCR) and the status (sensitive or resistant) of certaincell lines to Apo2L or DR5 antibody.

FIG. 11 provides a comparison of various colon or colorectal cancer celllines for expression of DcR1 or DcR2 receptors (as determined by FACS)and the status (sensitive or resistant) of certain cell lines to Apo2Lor DR5 antibody.

FIG. 12 show immunohistochemical staining for sialyl lewis A and X onfour colorectal cancer cell lines, CaCo2, SW 1417, DLD-1, and Colo 205,and its correlation to expression of sialyl Lewis A and X as measured byFACS and its correlation to sensitivity to Apo2L.

FIG. 13 shows a summary of IHC experiments demonstrating expression ofsialyl Lewis A and X in tissue samples of normal colon mucosa, normalliver tissue, primary colon cancer, and colon cancer metastases.

DETAILED DESCRIPTION OF THE INVENTION

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

Before the present methods and assays are described, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, animal species or genera,constructs, and reagents described as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “agenetic alteration” includes a plurality of such alterations andreference to “a probe” includes reference to one or more probes andequivalents thereof known to those skilled in the art, and so forth.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further the actualpublication dates may be different from those shown and requireindependent verification.

I. DEFINITIONS

The terms “Apo2L/TRAIL”, “Apo-2L”, and “TRAIL” are used herein to referto a polypeptide sequence which includes amino acid residues 114-281,inclusive, 95-281, inclusive, residues 92-281, inclusive, residues91-281, inclusive, residues 41-281, inclusive, residues 15-281,inclusive, or residues 1-281, inclusive, of the amino acid sequenceshown in FIG. 1, as well as biologically active fragments, deletional,insertional, or substitutional variants of the above sequences. In oneembodiment, the polypeptide sequence comprises residues 114-281 of FIG.1), and optionally, consists of residues 114-281 of FIG. 1. Optionally,the polypeptide sequence comprises residues 92-281 or residues 91-281 ofFIG. 1. The Apo-2L polypeptides may be encoded by the native nucleotidesequence shown in FIG. 1. Optionally, the codon which encodes residuePro119 (FIG. 1 may be “CCT” or “CCG”. In other embodiments, thefragments or variants are biologically active and have at least about80% amino acid sequence identity, more preferably at least about 90%sequence identity, and even more preferably, at least 95%, 96%, 97%,98%, or 99% sequence identity with any one of the above recitedApo2L/TRAIL sequences. Optionally, the Apo2L/TRAIL polypeptide isencoded by a nucleotide sequence which hybridizes under stringentconditions with the encoding polynucleotide sequence provided in FIG. 1.The definition encompasses substitutional variants of Apo2L/TRAIL inwhich at least one of its native amino acids are substituted by analanine residue. Particular substitutional variants of the Apo2L/TRAILinclude those in which at least one amino acid is substituted by analanine residue. These substitutional variants include those identified,for example, as “D203A”; “D218A” and “D269A.” This nomenclature is usedto identify Apo2L/TRAIL variants wherein the aspartic acid residues atpositions 203, 218, and/or 269 (using the numbering shown in FIG. 1) aresubstituted by alanine residues. Optionally, the Apo2L variants maycomprise one or more of the alanine substitutions which are recited inTable I of published PCT application WO 01/00832. Substitutionalvariants include one or more of the residue substitutions identified inTable I of WO 01/00832 published Jan. 4, 2001. The definition alsoencompasses a native sequence Apo2L/TRAIL isolated from an Apo2L/TRAILsource or prepared by recombinant or synthetic methods. The Apo2L/TRAILof the invention includes the polypeptides referred to as Apo2L/TRAIL orTRAIL disclosed in PCT Publication Nos. WO97/01633 and WO97/25428. Theterms “Apo2L/TRAIL” or “Apo2L” are used to refer generally to forms ofthe Apo2L/TRAIL which include monomer, dimer or trimer forms of thepolypeptide. All numbering of amino acid residues referred to in theApo2L sequence use the numbering according to FIG. 1, unlessspecifically stated otherwise. For instance, “D203” or “Asp203” refersto the aspartic acid residue at position 203 in the sequence provided inFIG. 1.

The term “Apo2L/TRAIL extracellular domain” or “Apo2L/TRAIL ECD” refersto a form of Apo2L/TRAIL which is essentially free of transmembrane andcytoplasmic domains. Ordinarily, the ECD will have less than 1% of suchtransmembrane and cytoplasmic domains, and preferably, will have lessthan 0.5% of such domains. It will be understood that any transmembranedomain(s) identified for the polypeptides of the present invention areidentified pursuant to criteria routinely employed in the art foridentifying that type of hydrophobic domain. The exact boundaries of atransmembrane domain may vary but most likely by no more than about 5amino acids at either end of the domain as initially identified. Inpreferred embodiments, the ECD will consist of a soluble, extracellulardomain sequence of the polypeptide which is free of the transmembraneand cytoplasmic or intracellular domains (and is not membrane bound).Particular extracellular domain sequences of Apo-2L/TRAIL are describedin PCT Publication Nos. WO97/01633 and WO97/25428.

The term “Apo2L/TRAIL monomer” or “Apo2L monomer” refers to a covalentchain of an extracellular domain sequence of Apo2L.

The term “Apo2L/TRAIL dimer” or “Apo2L dimer” refers to two Apo-2Lmonomers joined in a covalent linkage via a disulfide bond. The term asused herein includes free standing Apo2L dimers and Apo2L dimers thatare within trimeric forms of Apo2L (i.e., associated with another, thirdApo2L monomer).

The term “Apo2L/TRAIL trimer” or “Apo2L trimer” refers to three Apo2Lmonomers that are non-covalently associated.

The term “Apo2L/TRAIL aggregate” is used to refer to self-associatedhigher oligomeric forms of Apo2L/TRAIL, such as Apo2L/TRAIL trimers,which form, for instance, hexameric and nanomeric forms of Apo2L/TRAIL.Determination of the presence and quantity of Apo2L/TRAIL monomer,dimer, or trimer (or other aggregates) may be made using methods andassays known in the art (and using commercially available materials),such as native size exclusion HPLC (“SEC”), denaturing size exclusionusing sodium dodecyl sulphate (“SDS-SEC”), reverse phase HPLC andcapillary electrophoresis.

“Apo-2 ligand receptor” includes the receptors referred to in the art as“DR4” and “DR5” whose polynucleotide and polypeptide sequences are shownin FIGS. 2 and 3 respectively. Pan et al. have described the TNFreceptor family member referred to as “DR4” (Pan et al., Science,276:111-113 (1997); see also WO98/32856 published Jul. 30, 1998; WO99/37684 published Jul. 29, 1999; WO 00/73349 published Dec. 7, 2000;U.S. Pat. No. 6,433,147 issued Aug. 13, 2002; U.S. Pat. No. 6,461,823issued Oct. 8, 2002, and U.S. Pat. No. 6,342,383 issued Jan. 29, 2002).Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science,277:815-818 (1997) described another receptor for Apo2L/TRAIL (see also,WO98/51793 published Nov. 19, 1998; WO98/41629 published Sep. 24, 1998).This receptor is referred to as DR5 (the receptor has also beenalternatively referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8,TRICK2 or KILLER; Screaton et al., Curr. Biol., 7:693-696 (1997);Walczak et al., EMBO J., 16:5386-5387 (1997); Wu et al., NatureGenetics, 17:141-143 (1997); WO98/35986 published Aug. 20, 1998;EP870,827 published Oct. 14, 1998; WO98/46643 published Oct. 22, 1998;WO99/02653 published Jan. 21, 1999; WO99/09165 published Feb. 25, 1999;WO99/11791 published Mar. 11, 1999; US 2002/0072091 published Aug. 13,2002; US 2002/0098550 published Dec. 7, 2001; U.S. Pat. No. 6,313,269issued Dec. 6, 2001; US 2001/0010924 published Aug. 2, 2001; US2003/01255540 published Jul. 3, 2003; US 2002/0160446 published Oct. 31,2002, US 2002/0048785 published Apr. 25, 2002; U.S. Pat. No. 6,569,642issued May 27, 2003, U.S. Pat. No. 6,072,047 issued Jun. 6, 2000, U.S.Pat. No. 6,642,358 issued Nov. 4, 2003). As described above, otherreceptors for Apo-2L include DcR1, DcR2, and OPG (see, Sheridan et al.,supra; Marsters et al., supra; and Simonet et al., supra). The term“Apo-2L receptor” when used herein encompasses native sequence receptorand receptor variants. These terms encompass Apo-2L receptor expressedin a variety of mammals, including humans. Apo-2L receptor may beendogenously expressed as occurs naturally in a variety of human tissuelineages, or may be expressed by recombinant or synthetic methods. A“native sequence Apo-2L receptor” comprises a polypeptide having thesame amino acid sequence as an Apo-2L receptor derived from nature.Thus, a native sequence Apo-2L receptor can have the amino acid sequenceof naturally-occurring Apo-2L receptor from any mammal. Such nativesequence Apo-2L receptor can be isolated from nature or can be producedby recombinant or synthetic means. The term “native sequence Apo-2Lreceptor” specifically encompasses naturally-occurring truncated orsecreted forms of the receptor (e.g., a soluble form containing, forinstance, an extracellular domain sequence), naturally-occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants. Receptor variants may include fragments or deletionmutants of the native sequence Apo-2L receptor. FIG. 3A shows the 411amino acid sequence of human DR5 as published in WO 98/51793 on Nov. 19,1998. A transcriptional splice variant of human DR5 is known in the art.This DR5 splice variant encodes the 440 amino acid sequence of human DR5shown in FIGS. 3B and 3C as published in WO 98/35986 on Aug. 20, 1998.

“Death receptor antibody” is used herein to refer generally to antibodyor antibodies directed to a receptor in the tumor necrosis factorreceptor superfamily and containing a death domain capable of signallingapoptosis, and such antibodies include DR5 antibody and DR4 antibody.

“DR5 receptor antibody”, “DR5 antibody”, or “anti-DR5 antibody” is usedin a broad sense to refer to antibodies that bind to at least one formof a DR5 receptor or extracellular domain thereof. Optionally the DR5antibody is fused or linked to a heterologous sequence or molecule.Preferably the heterologous sequence allows or assists the antibody toform higher order or oligomeric complexes. Optionally, the DR5 antibodybinds to DR5 receptor but does not bind or cross-react with anyadditional Apo-2L receptor (e.g. DR4, DcR1, or DcR2). Optionally theantibody is an agonist of DR5 signalling activity.

Optionally, the DR5 antibody of the invention binds to a DR5 receptor ata concentration range of about 0.1 nM to about 20 mM as measured in aBIAcore binding assay. Optionally, the DR5 antibodies of the inventionexhibit an Ic 50 value of about 0.6 nM to about 18 mM as measured in aBIAcore binding assay.

“DR4 receptor antibody”, “DR4 antibody”, or “anti-DR4 antibody” is usedin a broad sense to refer to antibodies that bind to at least one formof a DR4 receptor or extracellular domain thereof. Optionally the DR4antibody is fused or linked to a heterologous sequence or molecule.Preferably the heterologous sequence allows or assists the antibody toform higher order or oligomeric complexes. Optionally, the DR4 antibodybinds to DR4 receptor but does not bind or cross-react with anyadditional Apo-2L receptor (e.g. DR5, DcR1, or DcR2). Optionally theantibody is an agonist of DR4 signalling activity.

Optionally, the DR4 antibody of the invention binds to a DR4 receptor ata concentration range of about 0.1 nM to about 20 mM as measured in aBIAcore binding assay. Optionally, the DR5 antibodies of the inventionexhibit an Ic 50 value of about 0.6 nM to about 18 mM as measured in aBIAcore binding assay.

The term “agonist” is used in the broadest sense, and includes anymolecule that partially or fully enhances, stimulates or activates oneor more biological activities of Apo2L/TRAIL, DR4 or DR5, in vitro, insitu, or in vivo. Examples of such biological activities binding ofApo2L/TRAIL to DR4 or DR5, include apoptosis as well as those furtherreported in the literature. An agonist may function in a direct orindirect manner. For instance, the agonist may function to partially orfully enhance, stimulate or activate one or more biological activitiesof DR4 or DR5, in vitro, in situ, or in vivo as a result of its directbinding to DR4 or DR5, which causes receptor activation or signaltransduction. The agonist may also function indirectly to partially orfully enhance, stimulate or activate one or more biological activitiesof DR4 or DR5, in vitro, in situ, or in vivo as a result of, e.g.,stimulating another effector molecule which then causes DR4 or DR5activation or signal transduction. It is contemplated that an agonistmay act as an enhancer molecule which functions indirectly to enhance orincrease DR4 or DR5 activation or activity. For instance, the agonistmay enhance activity of endogenous Apo-2L in a mammal. This could beaccomplished, for example, by pre-complexing DR4 or DR5 or bystabilizing complexes of the respective ligand with the DR4 or DR5receptor (such as stabilizing native complex formed between Apo-2L andDR4 or DR5).

The term “biomarker” as used in the present application refers generallyto a molecule, including a gene, protein, carbohydrate structure, orglycolipid, the expression of which in or on a mammalian tissue or cellcan be detected by standard methods (or methods disclosed herein) and ispredictive for a mammalian cell's or tissue's sensitivity to Apo2L/TRAILor death receptor antibody. Such biomarkers contemplated by the presentinvention include but are not limited to “(1,3/1,4) fucosyltransferase”or “FUT3”, “alpha (1,3) fucosyltransferase” or “FUT6”, “Sialyl Lewis A”,and “Sialyl Lewis X”. Optionally, the expression of such a biomarker isdetermined to be higher than that observed for a control tissue or cellsample. Optionally, for example, the expression of such a biomarker willbe determined in a PCR or FACS assay to be at least 50-fold, orpreferably at least 100-fold higher in the test tissue or cell samplethan that observed for a control tissue or cell sample. Optionally, theexpression of such a biomarker will be determined in an IHC assay toscore at least 2 or higher for staining intensity.

“(1,3/1,4) fucosyltransferase” or “FUT3” is used herein to refer to amolecule having structural features as described herein and optionally,catalyzing the transfer of a fucose residue from the donor substrate,GDP-fucose, to an acceptor substrate in an α3- or α4-linkage to GlcNAc(FUTs III-VII and IX). The DNA sequence and amino acid sequence forhuman FUT3 is provided in FIG. 4. These sequences correspond to GenBankAccession Number HSU27328 and are described for example inKukowska-Latallo et al., Genes Dev. 1990 August; 4 (8):1288-303. FUTsgenerally are type II transmembrane glycoproteins residing in the Golgivaccules, and typically composed of an N-terminal cytoplasmic tail, amembrane-spanning region, and a catalytic domain oriented lumenally inthe Golgi apparatus. Between the membrane-spanning region and thecatalytic domain is a region called the stem (Paulson and Colley, J.Biol. Chem., 264:17615-17618 (1989)).

“alpha (1,3) fucosyltransferase” or “FUT6” is used herein to refer to amolecule which structurally relates to, e.g, the DNA sequence and aminoacid sequence for human FUT6 provided in FIG. 5. These sequencescorrespond to GenBank Accession Number HSU27333 and are described forexample in Koszdin and Bowen, Biochem Biophys Res Commun. 1992 Aug. 31;187 (1):152-7. FUT 6 is typically expressed in epithelial cells and inliver, kidney, and gastrointestinal tissues, specifically, stomach,jejunum and colon (and typically minimally expressed in spleen, lung andcervix uteri). FUT 6 is typically not detected in brain, adrenal cortex,or peripheral blood leukocytes.

“Sialyl Lewis A” is used herein to refer to a tetrasaccharidecarbohydrate structure or antigen having the following sequence orstructure, which may be membrane bound or in soluble form, circulating,for instance, in serum:

“Sialyl Lewis X” is used herein to refer to tetrasaccharide carbohydratestructure or antigen having the following sequence or structure, whichmay be membrane bound or in soluble form, circulating, for instance, inserum:

By “subject” or “patient” is meant any single subject for which therapyis desired, including humans, cattle, dogs, guinea pigs, rabbits,chickens, insects and so on. Also intended to be included as a subjectare any subjects involved in clinical research trials not showing anyclinical sign of disease, or subjects involved in epidemiologicalstudies, or subjects used as controls.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

By “tissue or cell sample” is meant a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissueor cell sample may be solid tissue as from a fresh, frozen and/orpreserved organ or tissue sample or biopsy or aspirate; blood or anyblood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. The tissue sample mayalso be primary or cultured cells or cell lines. Optionally, the tissueor cell sample is obtained from a primary or metastatic tumor. Thetissue sample may contain compounds which are not naturally intermixedwith the tissue in nature such as preservatives, anticoagulants,buffers, fixatives, nutrients, antibiotics, or the like.

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention, provided that it is understood that the presentinvention comprises a method whereby the same section of tissue sampleis analyzed at both morphological and molecular levels, or is analyzedwith respect to both protein and nucleic acid.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis or protocol with theperformance and/or results of a second analysis or protocol. Forexample, one may use the results of a first analysis or protocol incarrying out a second protocols and/or one may use the results of afirst analysis or protocol to determine whether a second analysis orprotocol should be performed. With respect to the embodiment ofimmununohistochemical analysis or protocol one may use the results ofIHC to determine whether a specific therapeutic regimen should beperformed.

By “nucleic acid” is meant to include any DNA or RNA. For example,chromosomal, mitochondrial, viral and/or bacterial nucleic acid presentin tissue sample. The term “nucleic acid” encompasses either or bothstrands of a double stranded nucleic acid molecule and includes anyfragment or portion of an intact nucleic acid molecule.

By “gene” is meant any nucleic acid sequence or portion thereof with afunctional role in encoding or transcribing a protein or regulatingother gene expression. The gene may consist of all the nucleic acidsresponsible for encoding a functional protein or only a portion of thenucleic acids responsible for encoding or expressing a protein. Thenucleic acid sequence may contain a genetic abnormality within exons,introns, initiation or termination regions, promoter sequences, otherregulatory sequences or unique adjacent regions to the gene.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable or complementary determining regionsboth in the light chain and the heavy chain variable domains. The morehighly conserved portions of variable domains are called the frameworkregions (FRs). The variable domains of native heavy and light chainseach comprise four FRs, largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of, the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cell-mediatedcytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constantdomains that correspond to the different classes of antibodies arecalled α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (U.S. Pat. No.5,693,780).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmuse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

An antibody “which binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity and/or avidity such thatthe antibody is useful as a therapeutic or diagnostic agent fortargeting a cell expressing the antigen.

For the purposes herein, “immunotherapy” will refer to a method oftreating a mammal (preferably a human patient) with an antibody, whereinthe antibody may be an unconjugated or “naked” antibody, or the antibodymay be conjugated or fused with heterologous molecule(s) or agent(s),such as one or more cytotoxic agent(s), thereby generating an“immunoconjugate”.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antagonistor antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The expression “effective amount” refers to an amount of an agent (e.g.Apo2L/TRAIL, anti-DR4 or DR5 antibody etc.) which is effective forpreventing, ameliorating or treating the disease or condition inquestion.

The terms “treating”, “treatment” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.Consecutive treatment or administration refers to treatment on at leasta daily basis without interruption in treatment by one or more days.Intermittent treatment or administration, or treatment or administrationin an intermittent fashion, refers to treatment that is not consecutive,but rather cyclic in nature.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors; platelet-growth factor;transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-likegrowth factor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, and -gamma; colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-11, IL-12, IL-13, IL-17; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1,see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adriamycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimeterxate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including Nolvadex™), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston™); aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays (such as Alamar blueassays or MTT assays), FACS analysis, caspase activation, DNAfragmentation (see, for example, Nicoletti et al., J. Immunol. Methods,139:271-279 (1991), and poly-ADP ribose polymerase, “PARP”, cleavageassays known in the art.

As used herein, the term “disorder” in general refers to any conditionthat would benefit from treatment with the compositions describedherein, including any disease or disorder that can be treated byeffective amounts of Apo2L/TRAIL, an anti-DR4 antibody, and/or ananti-DR5 antibody. This includes chronic and acute disorders, as well asthose pathological conditions which predispose the mammal to thedisorder in question. Non-limiting examples of disorders to be treatedherein include benign and malignant cancers; inflammatory, angiogenic,and immunologic disorders, autoimmune disorders, arthritis (includingrheumatoid arthritis), multiple sclerosis, and HIV/AIDS.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. Moreparticular examples of such cancers include squamous cell carcinoma,myeloma, small-cell lung cancer, non-small cell lung cancer, glioma,gastrointestinal (tract) cancer, renal cancer, ovarian cancer, livercancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breastcancer, colon carcinoma, and head and neck cancer.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are autoimmune diseases, immune-mediated inflammatory diseases,non-immune-mediated inflammatory diseases, infectious diseases, andimmunodeficiency diseases. Examples of immune-related and inflammatorydiseases, some of which are immune or T cell mediated, which can betreated according to the invention include systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjogren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases such as inflammatory boweldisease (ulcerative colitis: Crohn's disease), gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis, allergic diseases such as asthma,allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includeAIDS (HIV infection), hepatitis A, B, C, D, and E, bacterial infections,fungal infections, protozoal infections and parasitic infections.

“Autoimmune disease” is used herein in a broad, general sense to referto disorders or conditions in mammals in which destruction of normal orhealthy tissue arises from humoral or cellular immune responses of theindividual mammal to his or her own tissue constituents. Examplesinclude, but are not limited to, lupus erythematous, thyroiditis,rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmunediabetes, and inflammatory bowel disease (IBD).

The term “tagged” when used herein refers to a chimeric moleculecomprising an antibody or polypeptide fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made or to provide some other function, such as theability to oligomerize (e.g. as occurs with peptides having leucinezipper domains), yet is short enough such that it generally does notinterfere with activity of the antibody or polypeptide. The tagpolypeptide preferably also is fairly unique so that a tag-specificantibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 to about 50 amino acid residues(preferably, between about 10 to about 20 residues).

The term “divalent metal ion” refers to a metal ion having two positivecharges. Examples of divalent metal ions include but are not limited tozinc, cobalt, nickel, cadmium, magnesium, and manganese. Particularforms of such metals that may be employed include salt forms (e.g.,pharmaceutically acceptable salt forms), such as chloride, acetate,carbonate, citrate and sulfate forms of the above mentioned divalentmetal ions. Optionally, a divalent metal ion for use in the presentinvention is zinc, and preferably, the salt form, zinc sulfate or zincchloride.

“Isolated,” when used to describe the various peptides or proteinsdisclosed herein, means peptide or protein that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepeptide or protein, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In preferred embodiments,the peptide or protein will be purified (1) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (2) to homogeneity bySDS-PAGE under non-reducing or reducing conditions using Coomassie blueor, preferably, silver stain, or (3) to homogeneity by massspectroscopic or peptide mapping techniques. Isolated material includespeptide or protein in situ within recombinant cells, since at least onecomponent of its natural environment will not be present. Ordinarily,however, isolated peptide or protein will be prepared by at least onepurification step.

“Percent (%) amino acid sequence identity” with respect to the sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art can determine appropriate parameters for measuringalignment, including assigning algorithms needed to achieve maximalalignment over the full-length sequences being compared. For purposesherein, percent amino acid identity values can be obtained using thesequence comparison computer program, ALIGN-2, which was authored byGenentech, Inc. and the source code of which has been filed with userdocumentation in the US Copyright Office, Washington, D.C., 20559,registered under the US Copyright Registration No. TXU510087. TheALIGN-2 program is publicly available through Genentech, Inc., South SanFrancisco, Calif. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA tore-anneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired identitybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“High stringency conditions”, as defined herein, are identified by thosethat: (1) employ low ionic strength and high temperature for washing;0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent;50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include overnight incubation at 37° C. ina solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

The term “primer” or “primers” refers to oligonucleotide sequences thathybridize to a complementary RNA or DNA target polynucleotide and serveas the starting points for the stepwise synthesis of a polynucleotidefrom mononucleotides by the action of a nucleotidyltransferase, asoccurs for example in a polymerase chain reaction.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and carry out ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and Fcγ RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seeDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)). FcRs herein includepolymorphisms such as the genetic dimorphism in the gene that encodesFcγRIIIa resulting in either a phenylalanine (F) or a valine (V) atamino acid position 158, located in the region of the receptor thatbinds to IgG1. The homozygous valine FcγRIIIa (FcγRIIIa-158V) has beenshown to have a higher affinity for human IgG1 and mediate increasedADCC in vitro relative to homozygous phenylalanine FcγRIIIa(FcγRIIIa-158F) or heterozygous (FcγRIIIa-158F/V) receptors.

“Complement dependent cytotoxicity” or “CDC” refer to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

II. TYPICAL METHODS AND MATERIALS OF THE INVENTION

The methods and assays disclosed herein are directed to the examinationof expression of one or more biomarkers in a mammalian tissue or cellsample, wherein the determination of that expression of one or more suchbiomarkers is predictive or indicative of whether the tissue or cellsample will be sensitive to apoptosis-inducing agents such asApo2L/TRAIL and anti-DR5 agonist antibodies. The methods and assaysinclude those which examine expression of biomarkers such as certainfucosyltransferases, in particular fucosyltransferase 3 (FUT3) and/orfucosyltransferase 6 (FUT6), as well as sialyl Lewis A and/or Xantigens.

As discussed above, there are some populations of diseased human celltypes (such as certain populations of cancer cells) which are resistantto apoptosis induction. It is therefore believed that the disclosedmethods and assays can provide for convenient, efficient, andpotentially cost-effective means to obtain data and information usefulin assessing appropriate or effective therapies for treating patients.For example, a patient having been diagnosed with cancer or an immunerelated condition could have a biopsy performed to obtain a tissue orcell sample, and the sample could be examined by way of various in vitroassays to determine whether the patient's cells would be sensitive to atherapeutic agent such as Apo2L/TRAIL or death receptor antibody.

The invention provides methods for predicting the sensitivity of amammalian tissue or cell sample (such as a cancer cell) to Apo2L/TRAILor a death receptor agonist antibody. In the methods, a mammalian tissueor cell sample is obtained and examined for expression of one or morebiomarkers. The methods may be conducted in a variety of assay formats,including assays detecting mRNA expression, enzymatic assays detectingpresence of enzymatic activity, and immunohistochemistry assays.Determination of expression of such biomarkers in said tissues or cellswill be predictive that such tissues or cells will be sensitive to theapoptosis-inducing activity of Apo2L/TRAIL and/or death receptorantibody. Applicants surprisingly found that the expression of suchparticular biomarkers correlates with the sensitivity of such tissuesand cells to apoptosis-inducing agents such as Apo2L/TRAIL and deathreceptor agonist antibodies.

As discussed below, expression of various biomarkers in a sample can beanalyzed by a number of methodologies, many of which are known in theart and understood by the skilled artisan, including but not limited to,immunohistochemical and/or Western analysis, quantitative blood basedassays (as for example Serum ELISA) (to examine, for example, levels ofprotein expression), biochemical enzymatic activity assays, in situhybridization, Northern analysis and/or PCR analysis of mRNAs, andgenomic Southern analysis (to examine, for example, gene deletion oramplification), as well as any one of the wide variety of assays thatcan be performed by gene and/or tissue array analysis. Typical protocolsfor evaluating the status of genes and gene products are found, forexample in Ausubel et al. eds., 1995, Current Protocols In MolecularBiology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15(Immunoblotting) and 18 (PCR Analysis).

The protocols below relating to detection of particular biomarkers, suchas fucosyltransferase 3 (FUT3), fucosyltransferase 6 (FUT6), SialylLewis A, and Sialyl Lewis X, in a sample are provided below forillustrative purposes.

* * *

Optional methods of the invention include protocols which examine ortest for presence of sialyl Lewis A and/or sialyl Lewis X proteins in amammalian tissue or cell sample. A variety of methods for detectingsialyl Lewis A and/or sialyl Lewis X-related protein can be employed andinclude, for example, immunohistochemical analysis, immunoprecipitation,Western blot analysis, molecular binding assays, ELISA, ELIFA,fluorescence activated cell sorting (FACS) and the like. For example, anoptional method of detecting the expression of sialyl Lewis A and/orsialyl Lewis X-related protein in a tissue or sample comprisescontacting the sample with a sialyl Lewis A and/or sialyl Lewis Xantibody, a sialyl Lewis A and/or sialyl Lewis X-reactive fragmentthereof, or a recombinant protein containing an antigen binding regionof a sialyl Lewis A and/or sialyl Lewis X antibody; and then detectingthe binding of sialyl Lewis A and/or sialyl Lewis X-related protein inthe sample.

In particular embodiments of the invention, the expression of sialylLewis A and/or sialyl Lewis X proteins in a sample is examined usingimmunohistochemistry and staining protocols. Immunohistochemicalstaining of tissue sections has been shown to be a reliable method ofassessing or detecting presence of proteins in a sample.Immunohistochemistry (“IHC”) techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods.

For sample preparation, a tissue or cell sample from a mammal (typicallya human patient) may be used. Examples of samples include, but are notlimited to, cancer cells such as colon, breast, prostate, ovary, lung,stomach, pancreas, lymphoma, and leukemia cancer cells. The sample canbe obtained by a variety of procedures known in the art including, butnot limited to surgical excision, aspiration or biopsy. The tissue maybe fresh or frozen. In one embodiment, the sample is fixed and embeddedin paraffin or the like.

The tissue sample may be fixed (i.e. preserved) by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology,” 3^(rd) edition (1960) Lee G. Luna,HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, NewYork; The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, ArmedForces Institute of Pathology, American Registry of Pathology,Washington, D.C.). One of skill in the art will appreciate that thechoice of a fixative is determined by the purpose for which the sampleis to be histologically stained or otherwise analyzed. One of skill inthe art will also appreciate that the length of fixation depends uponthe size of the tissue sample and the fixative used. By way of example,neutral buffered formalin, Bouin's or paraformaldehyde, may be used tofix a sample.

Generally, the sample is first fixed and is then dehydrated through anascending series of alcohols, infiltrated and embedded with paraffin orother sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra).Examples of paraffin that may be used include, but are not limited to,Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded,the sample may be sectioned by a microtome or the like (See e.g.,“Manual of Histological Staining Method of the Armed Forces Institute ofPathology”, supra). By way of example for this procedure, sections mayrange from about three microns to about five microns in thickness. Oncesectioned, the sections may be attached to slides by several standardmethods. Examples of slide adhesives include, but are not limited to,silane, gelatin, poly-L-lysine and the like. By way of example, theparaffin embedded sections may be attached to positively charged slidesand/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated to water. The tissuesections may be deparaffinized by several conventional standardmethodologies. For example, xylenes and a gradually descending series ofalcohols may be used (See e.g., “Manual of Histological Staining Methodof the Armed Forces Institute of Pathology”, supra). Alternatively,commercially available deparaffinizing non-organic agents such asHemo-De7 (CMS, Houston, Tex.) may be used.

Optionally, subsequent to the sample preparation, a tissue section maybe analyzed using IHC. IHC may be performed in combination withadditional techniques such as morphological staining and/or fluorescencein-situ hybridization. Two general methods of IHC are available; directand indirect assays. According to the first assay, binding of antibodyto the target antigen (e.g., sialyl Lewis A and/or sialyl Lewis X) isdetermined directly. This direct assay uses a labeled reagent, such as afluorescent tag or an enzyme-labeled primary antibody, which can bevisualized without further antibody interaction. In a typical indirectassay, unconjugated primary antibody binds to the antigen and then alabeled secondary antibody binds to the primary antibody. Where thesecondary antibody is conjugated to an enzymatic label, a chromogenic orfluorogenic substrate is added to provide visualization of the antigen.Signal amplification occurs because several secondary antibodies mayreact with different epitopes on the primary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Colloidal gold particles.

(c) Fluorescent labels including, but are not limited to, rare earthchelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl,Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commerciallyavailable fluorophores such SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/orderivatives of any one or more of the above. The fluorescent labels canbe conjugated to the antibody using the techniques disclosed in CurrentProtocols in Immunology, supra, for example. Fluorescence can bequantified using a fluorimeter.

(d) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed. J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g.,4-methylumbelliferyl-β-D-galactosidase).

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980. Sometimes, the label is indirectly conjugatedwith the antibody. The skilled artisan will be aware of varioustechniques for achieving this. For example, the antibody can beconjugated with biotin and any of the four broad categories of labelsmentioned above can be conjugated with avidin, or vice versa. Biotinbinds selectively to avidin and thus, the label can be conjugated withthe antibody in this indirect manner. Alternatively, to achieve indirectconjugation of the label with the antibody, the antibody is conjugatedwith a small hapten and one of the different types of labels mentionedabove is conjugated with an anti-hapten antibody. Thus, indirectconjugation of the label with the antibody can be achieved.

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired, For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong etal. Appl. Immunohistochem. 4 (3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation. The extent of binding ofantibody to the sample is determined by using any one of the detectablelabels discussed above. Preferably, the label is an enzymatic label(e.g. HRPO) which catalyzes a chemical alteration of the chromogenicsubstrate such as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g. the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

Optionally, the antibodies employed in the IHC analysis to detectexpression of sialyl Lewis A or anti-sialyl Lewis X, are anti-sialylLewis A and anti-sialyl Lewis X antibody, respectively. Optionally, theanti-sialyl Lewis A and the anti-sialyl Lewis X antibody is a monoclonalantibody. Anti-sialyl Lewis A and an anti-sialyl Lewis X antibodies arereadily available in the art, including from various commercial sources.

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope, and stainingintensity criteria, routinely used in the art, may be employed. Wherethe antigen is sialyl Lewis A and/or sialyl Lewis X protein, stainingintensity criteria may be evaluated as follows:

TABLE 1 Staining Pattern Score No staining is observed in cells. 0 Faint/barely perceptible staining is detected in 1+ more than 10% of thecells. Weak to moderate staining is observed in more 2+ than 10% of thecells. Moderate to strong staining is observed in more 3+ than 10% ofthe cells.

Typically, a staining pattern score of about 2+ or higher in such an IHCassay is believed to be predictive or indicative of sensitivity of amammalian cell (such as a mammalian cancer cell) to Apo2L/TRAIL or adeath receptor agonist antibody.

In alternative methods, the sample may be contacted with an antibodyspecific for said biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting said complex. Thepresence of the biomarker may be accomplished in a number of ways, suchas by Western blotting and ELISA procedures for assaying a wide varietyof tissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labelled antibodyto a target biomarker.

Sandwich assays are among the most useful and commonly used assays. Anumber of variations of the sandwich assay technique exist, and all areintended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized on a solidsubstrate, and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen complex, asecond antibody specific to the antigen, labelled with a reportermolecule capable of producing a detectable signal is then added andincubated, allowing time sufficient for the formation of another complexof antibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labelled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated for aperiod of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g. from room temperature to40° C. such as between 25° C. and 32° C. inclusive) to allow binding ofany subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the biomarker. The secondantibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the molecular marker.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibodywhich may or may not be labelled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labelling with the antibody.Alternatively, a second labelled antibody, specific to the firstantibody is exposed to the target-first antibody complex to form atarget-first antibody-second antibody tertiary complex. The complex isdetected by the signal emitted by the reporter molecule. By “reportermolecule”, as used in the present specification, is meant a moleculewhich, by its chemical nature, provides an analytically identifiablesignal which allows the detection of antigen-bound antibody. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, -galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody-molecular marker complex,allowed to bind, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of biomarker which was present in the sample.Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-molecular marker complex. After washingoff the unbound reagent, the remaining tertiary complex is then exposedto the light of the appropriate wavelength, the fluorescence observedindicates the presence of the molecular marker of interest.Immunofluorescence and EIA techniques are both very well established inthe art. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules, may also be employed.

It is contemplated that the above described techniques may also beemployed to detect expression of FUT3 or FUT 6 polypeptides.

Methods of the invention further include protocols which examine thepresence and/or expression of mRNAs, such as FUT3 and/or FUT6 mRNAs, ina tissue or cell sample. Methods for the evaluation of mRNAs in cellsare well known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeledFUT3 and/or FUT6 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for FUT3 and/or FUT6, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like).

Tissue or cell samples from mammals can be conveniently assayed for,e.g., FUT3 and/or FUT6 mRNAs using Northern, dot blot or PCR analysis.For example, RT-PCR assays such as quantitative PCR assays are wellknown in the art. In an illustrative embodiment of the invention, amethod for detecting an FUT3 and/or FUT6 mRNA in a biological samplecomprises producing cDNA from the sample by reverse transcription usingat least one primer; amplifying the cDNA so produced using an FUT3and/or FUT6 polynucleotide as sense and antisense primers to amplifyFUT3 and/or FUT6 cDNAs therein; and detecting the presence of theamplified FUT3 and/or FUT6 cDNA. In addition, such methods can includeone or more steps that allow one to determine the levels of FUT3 and/orFUT6 mRNA in a biological sample (e.g. by simultaneously examining thelevels a comparative control mRNA sequence of a “housekeeping” gene suchas an actin family member). Optionally, the sequence of the amplifiedFUT3 and/or FUT6 cDNA can be determined.

Material embodiments of this aspect of the invention include FUT3 and/orFUT6 primers and primer pairs, which allow the specific amplification ofthe polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of FUT3 and/or FUT6 polynucleotides in a sampleand as a means for detecting a cell expressing FUT3 and/or FUT6proteins. As will be understood by the skilled artisan, a great manydifferent primers and probes may be prepared based on the sequencesprovided in herein and used effectively to amplify, clone and/ordetermine the presence and/or levels of FUT3 and/or FUT6 mRNAs.

Optional methods of the invention include protocols which examine ordetect mRNAs, such as FUT3 and FUT6 or other fucosyltransferase mRNAs,in a tissue or cell sample by microarray technologies. Using nucleicacid microarrays, test and control mRNA samples from test and controltissue samples are reverse transcribed and labeled to generate cDNAprobes. The probes are then hybridized to an array of nucleic acidsimmobilized on a solid support. The array is configured such that thesequence and position of each member of the array is known. For example,a selection of genes that have potential to be expressed in certaindisease states may be arrayed on a solid support. Hybridization of alabeled probe with a particular array member indicates that the samplefrom which the probe was derived expresses that gene. Differential geneexpression analysis of disease tissue can provide valuable information.Microarray technology utilizes nucleic acid hybridization techniques andcomputing technology to evaluate the mRNA expression profile ofthousands of genes within a single experiment. (see, e.g., WO 01/75166published Oct. 11, 2001; (See, for example, U.S. Pat. Nos. 5,700,637,5,445,934, and 5,807,522, Lockart, Nature Biotechnology, 14:1675-1680(1996); Cheung, V. G. et al., Nature Genetics 21 (Suppl):15-19 (1999)for a discussion of array fabrication). DNA microarrays are miniaturearrays containing gene fragments that are either synthesized directlyonto or spotted onto glass or other substrates. Thousands of genes areusually represented in a single array. A typical microarray experimentinvolves the following steps: 1. preparation of fluorescently labeledtarget from RNA isolated from the sample, 2. hybridization of thelabeled target to the microarray, 3. washing, staining, and scanning ofthe array, 4. analysis of the scanned image and 5. generation of geneexpression profiles. Currently two main types of DNA microarrays arebeing used: oligonucleotide (usually 25 to 70 mers) arrays and geneexpression arrays containing PCR products prepared from cDNAs. Informing an array, oligonucleotides can be either prefabricated andspotted to the surface or directly synthesized on to the surface (insitu).

The Affymetrix GeneChip® system is a commercially available microarraysystem which comprises arrays fabricated by direct synthesis ofoligonucleotides on a glass surface. Probe/Gene Arrays:Oligonucleotides, usually 25 mers, are directly synthesized onto a glasswafer by a combination of semiconductor-based photolithography and solidphase chemical synthesis technologies. Each array contains up to 400,000different oligos and each oligo is present in millions of copies. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMicroarray Suite software. Each gene is represented on the array by aseries of different oligonucleotide probes. Each probe pair consists ofa perfect match oligonucleotide and a mismatch oligonucleotide. Theperfect match probe has a sequence exactly complimentary to theparticular gene and thus measures the expression of the gene. Themismatch probe differs from the perfect match probe by a single basesubstitution at the center base position, disturbing the binding of thetarget gene transcript. This helps to determine the background andnonspecific hybridization that contributes to the signal measured forthe perfect match oligo. The Microarray Suite software subtracts thehybridization intensities of the mismatch probes from those of theperfect match probes to determine the absolute or specific intensityvalue for each probe set. Probes are chosen based on current informationfrom Genbank and other nucleotide repositories. The sequences arebelieved to recognize unique regions of the 3′ end of the gene. AGeneChip Hybridization Oven (“rotisserie” oven) is used to carry out thehybridization of up to 64 arrays at one time. The fluidics stationperforms washing and staining of the probe arrays. It is completelyautomated and contains four modules, with each module holding one probearray. Each module is controlled independently through Microarray Suitesoftware using preprogrammed fluidics protocols. The scanner is aconfocal laser fluorescence scanner which measures fluorescenceintensity emitted by the labeled cRNA bound to the probe arrays. Thecomputer workstation with Microarray Suite software controls thefluidics station and the scanner. Microarray Suite software can controlup to eight fluidics stations using preprogrammed hybridization, wash,and stain protocols for the probe array. The software also acquires andconverts hybridization intensity data into a presence/absence call foreach gene using appropriate algorithms. Finally, the software detectschanges in gene expression between experiments by comparison analysisand formats the output into .txt files, which can be used with othersoftware programs for further data analysis.

The expression of a selected biomarker may also be assessed by examininggene deletion or gene amplification. Gene deletion or amplification maybe measured by any one of a wide variety of protocols known in the art,for example, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situhybridization (e.g., FISH), using an appropriately labeled probe,cytogenetic methods or comparative genomic hybridization (CGH) using anappropriately labeled probe. By way of example, these methods may beemployed to detect deletion of amplification of the FUT3 and/or FUT6genes.

Additionally, one can examine the methylation status of the biomarker,such as the FUT3 and/or FUT6 gene, in a tissue or cell sample. Aberrantdemethylation and/or hypermethylation of CpG islands in gene 5′regulatory regions frequently occurs in immortalized and transformedcells, and can result in altered expression of various genes. A varietyof assays for examining methylation status of a gene are well known inthe art. For example, one can utilize, in Southern hybridizationapproaches, methylation-sensitive restriction enzymes which cannotcleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995; De Marzo et al., Am. J. Pathol. 155 (6): 1985-1992(1999); Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998,7:531-536); and Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998).

Expression of a selected biomarker in a tissue or cell sample may alsobe examined by way of functional or activity-based assays. For instance,if the biomarker is an enzyme, one may conduct assays known in the artto determine or detect the presence of the given enzymatic activity inthe tissue or cell sample.

In the methods of the present invention, it is contemplated that thetissue or cell sample may also be examined for the expression ofApo2L/TRAIL or receptors in the sample which bind Apo2L/TRAIL or deathreceptor antibody. As described above and in the art, it is presentlybelieved Apo2L/TRAIL binds to at least five different receptors: DR4,DR5, DcR1, DcR2, and OPG. Using methods known in the art, includingthose described herein, the expression of Apo2L/TRAIL, DR4, DR5, DcR1,DcR2 and/or OPG can be detected on the mRNA level and on the proteinlevel. As shown in FIGS. 10 and 11, data suggests that examining thetissue or cell sample for expression of DcR1 and/or DcR2 receptors maygive further information as to whether the tissue or cell sample will besensitive to either Apo2L/TRAIL or death receptor antibody. By way ofexample, the IHC techniques described above may be employed to detectthe presence of one of more such molecules in the sample. It iscontemplated that in methods in which a tissue or sample is beingexamined not only for the presence of a FUT or Lewis antigen marker, butalso for the presence, e.g., DR4, DR5 or DcR1, separate slides may beprepared from the same tissue or sample, and each slide tested with areagent specific for each specific biomarker or receptor. Alternatively,a single slide may be prepared from the tissue or cell sample, andantibodies directed to each biomarker or receptor may be used inconnection with a multi-color staining protocol to allow visualizationand detection of the respective biomarkers or receptors.

Subsequent to the determination that the tissue or cell sample expressesone or more of the biomarkers indicating the tissue or cell sample willbe sensitive to the activity of Apo2L/TRAIL or death receptor antibody,it is contemplated that an effective amount of the Apo2L/TRAIL or deathreceptor antibody may be administered to the mammal to treat a disorder,such as cancer or immune related disorder which is afflicting themammal. Diagnosis in mammals of the various pathological conditionsdescribed herein can be made by the skilled practitioner. Diagnostictechniques are available in the art which allow, e.g., for the diagnosisor detection of cancer or immune related disease in a mammal. Forinstance, cancers may be identified through techniques, including butnot limited to, palpation, blood analysis, x-ray, NMR and the like.Immune related diseases can also be readily identified.

The Apo2L/TRAIL or death receptor antibody can be administered in accordwith known methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Optionally, administration may be performed through mini-pump infusionusing various commercially available devices.

Effective dosages and schedules for administering Apo2L/TRAIL or deathreceptor antibody may be determined empirically, and making suchdeterminations is within the skill in the art. Single or multipledosages may be employed. It is presently believed that an effectivedosage or amount of Apo2L/TRAIL used alone may range from about 1 μg/kgto about 100 mg/kg of body weight or more per day. Interspecies scalingof dosages can be performed in a manner known in the art, e.g., asdisclosed in Mordenti et al., Pharmaceut. Res., 8:1351 (1991).

When in vivo administration of Apo2L/TRAIL is employed, normal dosageamounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal bodyweight or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day,depending upon the route of administration. Guidance as to particulardosages and methods of delivery is provided in the literature; see, forexample, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It isanticipated that different formulations will be effective for differenttreatment compounds and different disorders, that administrationtargeting one organ or tissue, for example, may necessitate delivery ina manner different from that to another organ or tissue.

It is contemplated that yet additional therapies may be employed in themethods. The one or more other therapies may include but are not limitedto, administration of radiation therapy, cytokine(s), growth inhibitoryagent(s), chemotherapeutic agent(s), cytotoxic agent(s), tyrosine kinaseinhibitors, ras farnesyl transferase inhibitors, angiogenesisinhibitors, and cyclin-dependent kinase inhibitors which are known inthe art and defined further with particularity above. It is contemplatedthat such other therapies may be employed as an agent separate from theApo2L/TRAIL or death receptor antibody. In addition, therapies based ontherapeutic antibodies that target tumor antigens such as Rituxan™ orHerceptin™ as well as anti-angiogenic antibodies such as anti-VEGF.

Preparation and dosing schedules for chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeuticagent may precede, or follow administration of the Apo2L/TRAIL or deathreceptor antibody, or may be given simultaneously therewith.

It may be desirable to also administer antibodies against otherantigens, such as antibodies which bind to CD20, CD11a, CD18, CD40,ErbB2, EGFR, ErbB3, ErbB4, vascular endothelial factor (VEGF), or otherTNFR family members (such as OPG, TNFR1, TNFR2, GITR, Apo-3, TACI, BCMA,BR3). Alternatively, or in addition, two or more antibodies binding thesame or two or more different antigens disclosed herein may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. Followingadministration, treated cells in vitro can be analyzed. Where there hasbeen in vivo treatment, a treated mammal can be monitored in variousways well known to the skilled practitioner. For instance, tumor cellscan be examined pathologically to assay for necrosis or serum can beanalyzed for immune system responses.

For use in the applications described or suggested above, kits orarticles of manufacture are also provided by the invention. Such kitsmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a FUT3 and/orFUT6 protein or a FUT3 and/or FUT6 gene or message, respectively. Wherethe kit utilizes nucleic acid hybridization to detect the target nucleicacid, the kit may also have containers containing nucleotide(s) foramplification of the target nucleic acid sequence and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic, florescent, or radioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The kits of the invention have a number of embodiments. A typicalembodiment is a kit comprising a container, a label on said container,and a composition contained within said container; wherein thecomposition includes a primary antibody that binds to a FUT3 and/or FUT6polypeptide sequence, the label on said container indicates that thecomposition can be used to evaluate the presence of FUT3 and/or FUT6proteins in at least one type of mammalian cell, and instructions forusing the FUT3 and/or FUT6 antibody for evaluating the presence of FUT3and/or FUT6 proteins in at least one type of mammalian cell. The kit canfurther comprise a set of instructions and materials for preparing atissue sample and applying antibody and probe to the same section of atissue sample. The kit may include both a primary and secondaryantibody, wherein the secondary antibody is conjugated to a label, e.g.,an enzymatic label.

Another embodiment is a kit comprising a container, a label on saidcontainer, and a composition contained within said container; whereinthe composition includes a polynucleotide that hybridizes to acomplement of the FUT3 and/or FUT6 polynucleotide under stringentconditions, the label on said container indicates that the compositioncan be used to evaluate the presence of FUT3 and/or FUT6 in at least onetype of mammalian cell, and instructions for using the FUT3 and/or FUT6polynucleotide for evaluating the presence of FUT3 and/or FUT6 RNA orDNA in at least one type of mammalian cell.

Other optional components in the kit include one or more buffers (e.g.,block buffer, wash buffer, substrate buffer, etc), other reagents suchas substrate (e.g., chromogen) which is chemically altered by anenzymatic label, epitope retrieval solution, control samples (positiveand/or negative controls), control slide(s) etc.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the examples that follow, none of which are intended to limitthe scope of the invention.

Methods and Materials:

Cell Culture and Cell Lines.

The following human colorectal adenocarcinoma cell lines: HCT-8, COLO205, HCT 116, SW403, LoVo, SW948, Caco-2, COLO 201, SW1417, DLD-1, CX-1,HCT-15, LS 180, RKO, RKO-AS45-1, SK-CO-1, SW480, SW620, SW837, CL-40,COLO-206F, COLO 320DM, COLO 320HSR, COLO-678, HT-29, KM12, LS1034,SW1116 were obtained from ATCC Depository (Manassas, Va.), DSMZ (GermanCollection of Microorganisms and Cell Cultures), JCRB (Japanese CellResources Bank) or ECACC (European Collection of Cell Cultures) andcultured in RPMI-1640 media supplemented with 10% heat inactivated fetalbovine serum, 2 mM L-glutamine and 10 mM HEPES.

Cytotoxicity Assays.

The MTT assay (CellTiter 96® Non-Radioactive Cell Proliferation Assayfrom Promega), which is a colorimetric assay based on the ability ofviable cells to reduce a soluble yellow tetrazolium salt (MTT) to blueformazan crystals), was used to determine the amount of viable cellsafter treatment with Apo2L/TRAIL or DR5 antibody. The MTT assay wasperformed by the addition of a premixed optimized dye solution toculture wells of a 96-well plate containing various concentrations (0 to1000 ng/ml) of Apo2L/TRAIL or DR5 antibody. During a 4-hour incubation,living cells convert the tetrazolium component of the dye solution intoa formazan product. The solubilization/stop solution was then added tothe culture wells to solubilize the formazan product, and the absorbanceat 570 nm was recorded using a 96-well plate reader (SpectraMax). The570 nm absorbance reading is directly proportional to the number ofcells normally used in proliferation assays. Although the absorbancemaximum for the formazan product is 570 nm and pure solutions appearblue, the color at the end of the assay may not be blue and depends onthe quantity of formazan present relative to other components (includingserum, acidified phenol red and unreduced MTT) in the culture medium.

Cell numbers were optimized by performing a cell titration to produce anassay signal near the high end of the linear range of the assay. Sincedifferent cell types have different levels of metabolic activity, thiswas done for each cell line separately. For most tumor cells examined,5,000 cells per well to 20,000 cells per well were used.

The following is a step by step description of the assays employed:

-   1. Cells used for bioassay were from stock cultures.-   2. Determination of cell number and trypan blue viability, and    suspension of the cells to a final number of 5,000 to 20,000 cells    per well.-   3. Dispensed 50 μl of the cell suspension into 96-well plate.-   4. Incubation of the plates at 37° C. in a humidified 5% CO₂    atmosphere over night.-   5. Addition of 50 μl culture medium containing various    concentrations ranging from 0 to 1000 ng/ml of Apo2L/TRAIL or DR5    antibody to samples of the 96-well plate. The controls were 50 μl of    culture medium (without Apo2L/TRAIL or DR5 antibody) and 100 μl    culture medium (without cells). Every experiment was performed in a    triplicate set of wells and at three independent days. The total    volume of the wells was 100 μl/well.-   6. Incubation of the plates at 37° C. for 72 hours in a humidified    5% CO₂ atmosphere.-   7. Addition of 15 μl of dye solution to each well.-   8. Incubation of the plates at 37° C. for up to 4 hours in a    humidified 5% CO₂ atmosphere.-   9. Addition of 100 μl of the solubilization/stop solution to each    well.-   10. Incubation of the plates overnight at 37° C. overnight.-   11. Record the absorbance at 570 nm wavelength using a 96-well plate    reader. A reference wavelength of 750 nm was used to reduce    background contributed by cell debris, fingerprints and other    nonspecific absorbance.-   12. The average of the absorbance values for the negative control    was used as a blank value and subtracted from all other readings.    The average of the absorbance values for each concentration of    Apo2L/TRAIL or DR5 antibody was divided by the average of the    absorbance values of the positive control (100% viable    cells−untreated) to calculate the amount of viable cells (in %).-   13. Percent viable cells (Y axis) versus concentration of    Apo2L/TRAIL or DR5 antibody (X axis, log scale) was plotted and the    IC50 value was determined by locating the X-axis value (ng/ml)    corresponding to 50% viable cells.    Affymetrix Labeling Protocol

An OD260/280 reading was taken for all samples, and samples were run onthe BioAnalyzer. 5 μg high quality Total RNA was used.

A. First Strand cDNA Synthesis:

1. Primer Hybridization

DEPC-H2O x μl Mix by vortexing. Quick spin. RNA (5 ug) y μl Incubate at70° C. for 10 minutes. Spike (1:4 dil of stock 1 μl Quick spin and puton ice for 5 ug) T7-(dT) 24 primer 1 μl volume 12 μl2. Temperature Adjustment

5X-1st strand and cDNA buffer 4 μl Add 7 μl (of the mix to the left) toeach sample. 0.1 M DTT 2 μl Mix by vortexing. Quick spin. 10 mM dNTP mix1 μl Incubate at 42° C. for 2 minutes. volume 7 μl3. First Strand Synthesis

Add 1 μl SSII RT to each sample.

SSII RT  1 μl Mix by pipetting up and down -OR- vortex lightly. Quickspin. Total volume 20 μl Incubate at 42° C. for 1 hour.B. Second Strand cDNA Synthesis

-   1. Place First Strand reactions on ice. Centrifuge briefly to bring    down condensation on sides of tube.-   2. Make the following Second strand master-mix.

DEPC-treated H2O 90 μl  5X-2nd Strand Reaction Buffer 30 μl  10 mM dNTPmix 3 μl 10 U/μl DNA Ligase 1 μl 10 U/μl DNA Polymerase I 4 μl  2 U/μlRNase H 1 μl Total volume 130 μl  

-   3. Add 130 μl Second strand master-mix to the 20 μl First strand    cDNA. (Final volume=150 μl)-   4. Mix by pipetting up and down—OR—by vortexing lightly. Quick spin.-   5. Incubate at 16° C. for 2 hours in a cooling water bath.-   6. Add 2 μl [10 U] T4 DNA Polymerase. Mix by pipetting up and    down—OR—vortex lightly. Quick spin.-   7. Incubate for 5 minutes at 16° C.-   8. Add 10 μl 0.5 M EDTA. Vortex lightly. Quick spin.-   9. Proceed to cleanup procedure for cDNA—OR—store at −20° C. for    later use.    Cleanup of Double-Stranded cDNA (GeneChip Sample Cleanup Module)-   1. Add 600 μl cDNA Binding Buffer to the 162 μl final    double-stranded cDNA synthesis preparation.

Mix by vortexing for 3 seconds.

-   2. Check that the color of the mixture is yellow (similar to cDNA    Binding Buffer w/o the cDNA synthesis reaction).

If the color of the mixture is orange or violet, add 10 μl of 3 M sodiumacetate, pH5.0 and mix.

The color of the mixture will turn to yellow.

-   3. Apply 500 μl of the sample to the cDNA Cleanup Spin Column    sitting in a 2 ml Collection Tube, and centrifuge

for 1 minute at ≧8,000×g (≧10,000 rpm). Discard flow-through ashazardous waste.

-   4. Reload the spin column with the remaining mixture (262 μl) and    centrifuge as above.

Discard flow-through as hazardous waste and discard the Collection Tube.

-   5. Transfer spin column into a new 2 ml Collection Tube (supplied).    Pipet 750 μl cDNA Wash Buffer onto the spin column. Centrifuge for 1    minute at 8,000×g (≧10,000 rpm).

Discard flow-through.

-   6. Open the cap of the spin column and centrifuge for 5 minutes at    maximum speed (≦25,000×g). Place

columns into the centrifuge using every second bucket. Position capsover the adjoining bucket so that

they are oriented in the opposite direction to the rotation, i.e., ifrotation is clockwise, orient caps

in a counter-clockwise direction. This avoids damage to caps.

Discard flow-through and Collection Tube.

-   7. Transfer spin column into a 1.5 ml Collection Tube. Pipet 10 μl    of cDNA Elution Buffer directly onto the spin

column membrane. Ensure that the cDNA Elution buffer is dispenseddirectly onto the membrane.

Incubate for 1 minute at room temperature and centrifuge 1 minute atmax. speed (≦25,000×g) to elute.

Setting Up and Running the IVT Reaction

Enzo: Bioarray HighYield RNA transcript Labeling Kit (Part No. 900182)

-   1. Use 10 μl of the Cleaned-up Double-stranded cDNA-   2. Make the following IVT master-mix:

Distilled or Deionized H2O 12 μl  10X HY Reaction buffer 4 μl 10X Biotinlabeled Ribonucleotides 4 μl 10X DTT 4 μl 10X RNase Inhibitor Mix 4 μl20X T7 RNA Polymerase 2 μl Total volume: 30 μl 

-   3. Add 30 μl of the IVT master-mix to 10 μl double-stranded cDNA.    (Total volume=40 μl)-   4. Mix by pipetting up and down—OR—by vortexing lightly. Quick spin.-   5. Immediately place the tube in a 37° C. water bath. Incubate for 5    hours.-   6. Store at −20° C. if not purifying RNA immediately.    Cleanup of Biotin-Labeled cRNA (GeneChip Sample Cleanup Module)-   1. Add 60 μl H2O to the IVT reaction and mix by vortexing for 3    seconds.-   2. Add 350 μl IVT cRNA Binding Buffer to the sample, mix by    vortexing for 3 seconds.-   3. Add 250 μl ethanol (96-100%) to the lysate, and mix well by    pipetting. Do not centrifuge.-   4. Apply sample (700 μl) to the IVT cRNA Cleanup Spin Column sitting    in a 2 ml collection tube.

Centrifuge for 15 seconds at ≧8,000×g (≧10,000 rpm).

-   5. Pass the eluate through the column once more.

Centrifuge for 15 seconds at ≧8,000×g (≧10,000 rpm).

Discard the flow-through as hazardous waste and discard the collectiontube.

-   6. Transfer the spin column into a new 2-ml collection tube    (supplied).-   7. Add 500 μl IVT cRNA Wash Buffer and centrifuge for 15 seconds at    ≧8,000×g (10,000 rpm) to wash.

Discard the flow-through.

-   8. Pipet 500 μl 80% (v/v) ethanol onto the spin column, and    centrifuge for 15 seconds at

≧8,000×g (≧10,000 rpm). Discard flow-though.

-   9. Open the cap of the spin column and centrifuge for 5 minutes at    max. speed (≦25,000×g).

Discard flow-through and Collection Tube.

-   10. Transfer the spin column into a new 1.5 ml collection tube.-   11. Pipet 11 μl RNase-free water directly onto the spin column    membrane. Let stand for 1 minute.

Centrifuge for 1 minute at maximum speed (≦25,000×g) to elute.

-   12. Pipet 10 μl RNase-free water directly onto the spin column    membrane. Let stand for 1 minute.

Centrifuge for 1 minute at maximum speed (≦25,000×g) to elute.

Quantifying the cRNA (IVT Product)

Use spectrophotometric analysis to determine the RNA yield. Apply theconvention that 1 OD at 260 nm equals

40 μg/ml RNA.

Check the OD at 260 nm and 280 nm to determine sample concentration andpurity.

Maintain the A260/A280 ratio close to 2.0 for pure RNA (ratios between1.9 and 2.1 are acceptable).

For quantification of cRNA when using total RNA as starting material, anadjusted cRNA yield must be calculated to

reflect carryover of unlabeled total RNA. Using an estimate of 100%carryover, use the formula below to

determine adjusted cRNA yield:adjusted cRNA yield=RNAm−(total RNAi)(y)

RNAm=amount of cRNA measured after IVT (μg)

total RNAi=starting amount of total RNA (μg)

y=fraction of cDNA reaction used in IVT

Fragmenting the cRNA for Target Preparation

For fragmentation, use the adjusted cRNA concentration.

-   1. Add 2 μl of 5× Fragmentation Buffer for every 8 μl of RNA plus    H2O.

20 μg CRNA 1 to 32 μl 5X Fragmentation Buffer 8 μl RNase-free water to40 μl Total volume: 40 μl

-   2. Incubate at 94° C. for 30 minutes. Immediately, put on ice    following the incubation.    Preparing the Hybridization Target-   1. Heat the 20× Eukaryotic Hybridization Controls and the Oligo B2    for 5 minutes at 65° C.

Affymetrix GeneChip Eukaryotic Hybridization Control Kit, Part #900362(for 150 rxns)

-   2. Lightly vortex, spin down.-   3. Master mix (Assuming the fragmented cRNA concentration is 0.5    μg/μl):

Standard Array (μl) Final Conc. Fragmented cRNA 15 ug 30 0.05 μg/μlOligo B2 (3 nM) 5 50 pM 20x Control Spike 15 1.5, 5, 25, 100 pM (Bio B,C, D, Cre) Herring Sperm DNA 3 0.1 mg/ml Acetylated BSA 3 0.5 mg/ml Hucot-1 DNA (1 mg/ml) 30 0.1 mg/ml 2X MES Hyb Buffer 150 1X H2O 64 FinalVolume 300

-   4. Aliquot 270 μl master mix into tubes and add 30 μl of fragmented    cRNA to each. This is the Hybridization Mix.-   5. Equilibrate the probe arrays to room temperature immediately    before use.-   6. Fill the probe array with 1×MES Hyb Buffer, and incubate in the    rotisserie oven for 10 minutes at 45° C., 60 rpm.-   7. Heat the Hybridization Mix in a 99° C. waterbath for 5 minutes.-   8. Transfer the Hybridization Mix to a 45° C. waterbath for 5    minutes.-   9. Centrifuge the Hybridization Mix for 5 minutes at maximum speed.-   10. Remove the 1×MES Hyb Buffer from the probe arrays.-   11. Fill the probe array with the top 200 μl of the Hybridization    Mix.-   12. Seal the septa with Tough-Spots.-   13. Hybridize the probe array at 45° C., 60 RPM for 19 hours.-   14. Wash, stain and scan the probe array according to the Affymetrix    protocols.    Affymetrix Materials

Item Vendor Catalog # T7-(dT)24 primer Biosearch custom TechnolgiesControl spikes in-house — Superscript II/5X First Strand Invitrogen18064-014 Buffer/0.1 M DTT 5X Second Strand Buffer Invitrogen 10812-01410 mM dNTP Invitrogen 18427-088 10 U/ul E. coli DNA Invitrogen 18052-019Ligase 10 U/ul E. coli DNA Invitrogen 18010-025 Polymerase I 2 U/ulRNase H Invitrogen 18021-071 10 U/ul T4 DNA Polymerase Invitrogen18005-025 0.5 M EDTA Sigma E-7889 ENZO High Yield RNA TranscriptAffymetrix 900182 labeling kit or ENZO (ENZO) GeneChip Sample CleanupModule Affymetrix 900371 Acetylated Bovine Serum Albumin Invitrogen15561-020 Goat IgG - Reagent Grade Sigma I-5256 Anti-streptavidinantibody (goat), Vector Labs BA-0500 biotinylated R-PhycoerythrinStreptavidin Molecular Probes S-866 20X SSPE BioWhittaker 51214Eukaryotic Control Kit Affymetrix 900362 Water, Molecular Biology GradeAmbion 9934 Human Cot-1 DNA Roche 1-581-074 5 M NaCl RNase-free,DNase-free Ambion 9760 Antifoam 0-30 Sigma A-8082 10% Tween-20 PierceChemical 28320 MES Free Acid Monohydrate Sigma M5287 MES Sodium SaltSigma M3885 EDTA Disodium Salt, 0.5 M solution Sigma E7889 Tough Spots,Label Dots USA Scientific 9902 GeneChip Hybridization Oven 640Affymetrix 800139 GeneChip Scanner 3000 w/Workstation Affymetrix 00-0074Fluidics Station Affymetrix 00-0081 Autoloader w/External Barcode ReaderAffymetrix 00-0129Quantitative PCRcDNA Synthesis:

Component Volume (uL) 10X RT Buffer 10 25X dNTP mixture 4 10X RandomPrimers 10 MultiScribe RT 5 (50 U/uL) RNase-free H2O 21 RNA (100 ng) 50Final Volume 100Incubation Conditions:

-   25° for 10 minutes-   37° for 2 hours    Taqman Reaction Using the ABI Prism 7700 Sequencing Detector:

Component Volume (uL) TaqMan Universal PCR 25 Master Mix (2X) TaqManprobe (20X) 2.5 (Assays-on-Demand ™) cDNA (100 ng) 2 H2O 20.5 FinalVolume 50Thermal Cycling Conditions:

-   95° for 10 minutes-   40 cycles: 95° for 15 seconds    -   60° for 1 minute    -   TaqMan probes: Assays-on-Demand™ (TaqMan® MGB probes, FAM™        dye-labeled)    -   Amplification of the endogenous control, GAPDH (probe        concentration 100 nM, forward & reverse primer concentrations        200 nM), was performed to standardize the amount of sample RNA        (cDNA) added to each reaction.        Relative quantitation was performed using the standard curve        method. For quantitation normalized to an endogenous control,        standard curves were prepared for both the target and the        endogenous reference. For each experimental sample, the amount        of target and endogenous reference was determined from the        appropriate standard curve. Then, the target amount was divided        by the endogenous reference amount to obtain a normalized target        value. One of the experimental samples served as the calibrator,        or 1× sample. Each of the normalized target values was then        divided by the calibrator normalized target value to generate        the relative expression levels.        FACS/Flow Cytometry (2° Antibody Staining Protocol):        All incubations and spins were performed at 4° C. and the tubes        kept on ice while not in the refrigerator.-   1. Determine the tube format by identifying the cell lines to be    used, the antibodies of interest, and any special conditions or    treatments.    -   a. controls.        -   i. Unstained, 2° Antibody, and compensation if the            fluorochromes have overlapping emission spectra.    -   b. Example:

Time Tube Cell Line (min) 1° Antibody 2° Antibody 1 e.g., COLO- 0 — —205 2 e.g., COLO- 0 — anti-Mouse- 205 FITC 3 e.g., COLO- 0 anti-SialylLewis A anti-Mouse- 205 FITC 4 e.g., COLO- 0 anti-CD15s (Sialylanti-Mouse- 205 Lewis X) FITC

-   2. Label the FACS tubes.    -   a. BD Falcon 12×75 mm Polystyrene Round-Bottom. Catalog #:        352052-   3. Prepare the cells for staining.    -   a. Treat adherent cells with Accutase or Trypsin.        -   i. Innovative Cell Technologies Inc, Accutase.        -   ii. Gibco, Trypsin. Catalog #: 25200-106.    -   b. Proceed with the remaining steps if the cells are suspension.-   4. Aliquot the cells into a 15 mL or 50 mL conical tube.-   5. Spin the cells for 5 min, 1200 rpm, 4° C.-   6. Aspirate the supernatant.-   7. Resuspend the cells in 5 mLs FACS Buffer.-   8. Spin the cells for 5 min, 1200 rpm, 4° C.-   9. Aspirate the supernatant.-   10. Resuspend the cells in Blocking Buffer.    -   a. Determine the volume of blocking buffer needed:        -   i. Number of tubes per cell line/treatment×100 μl Blocking            Buffer per tube.        -   ii. Want 1×10⁶ cells per 100 μl of Blocking Buffer.-   11. Aliquot 100 μl of the cells into the appropriate tube.    -   a. Based on the pre-determined tube format.-   12. Add the 1° antibody to the appropriate tube.    -   a. Lewis A:        -   i. Use 10 μl of 0.2 μg/μl stock per tube.            -   1. Final concentration is 2 μg.        -   ii. Chemicon: anti-Sialyl Lewis A. Catalog #: MAB2095.    -   b. Lewis X:        -   i. Use 5 μl of 0.5 μg/μl stock per tube.            -   1. Final concentration is 2.5 μg.        -   ii. BD Pharmingen: CD15s (Sialyl Lewis X). Catalog #:            551344.-   13. Incubate for 30 min at 4° C.-   14. Add 1 mL of FACS Buffer to each tube.-   15. Spin the cells for 5 min, 1200 rpm, 4° C.-   16. Aspirate the supernatant-   17. Gently rack the tubes to dislodge the pellet.    -   a. “Rack”—Run the tubes across the surface of the 12×75 mm tube        rack.-   18. Repeat steps 14-17.-   19. Add 100 μl of Blocking Buffer to each tube.-   20. Add the 2° antibody into the appropriate tube.    -   a. Use 10 μl per tube.    -   b. Jackson, Goat-anti-Mouse FITC. Catalog #: 115-096-068.-   21. Incubate for 30 min at 4° C.-   22. Repeat steps 14-17 twice.-   23. Resuspend cells in FACS Buffer/PI.    -   a. Determine volume needed:        -   i. Need 1 mL of solution per tube.        -   ii. PI=1 μl per 1 mL of Buffer.    -   b. Molecular Probes, Propidium Iodide. Catalog 4: P3566.-   24. Place tubes in an ice bucket or iced tube rack.-   25. Cover with aluminum foil and take to the FACS lab for a    qualified operator to acquire and analyze samples.    5% Blocking Buffer:

1. FBS to 5% of total volume.

2. FACS Buffer.

3. Filter the solution through a 0.2 μm filter.

FACS Buffer:

1. 980 mLs PBS.

2. 8 mLs 0.25M EDTA.

3. 20 mLs FBS.

4. Filter the solution through a 0.2 μm filter.

Immunohistochemistry Procedure: Sialyl Lewis A

Antibody: Sialyl Lewis A AB-1

Clone: 121SLE

Supplier: NeoMarkers

Catalog No. MS-279-P

Ig Species Mouse

IHC Method: Paraffin

Pretreatment: None

IHC Handling: Autostainer

Isotype: Mouse IgM

Procedure Species: Human

IgG Concentration: 200 ug/ml

Normal Procedure:

-   Deparaffinize and hydrate to distilled water.-   Block endogenous biotin with Vector Avidin Biotin Blocking System.-   Rinse with TBS: 2 changes, 5 minutes each.-   Block with 10% Normal Horse Serum for 30 minutes at RT.-   Incubate sections with Mouse Monoclonal Sialyl Lewis A antibody    diluted to 5 ug/ml with 10% normal Horse Serum for 60 minutes at RT.    -   Use a mouse isotype IgM diluted at 5 ug/ml in 10% Normal Horse        Serum for the negative control.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with biotinylated horse anti-mouse antibody; 1:200    diluted in 10% Normal Horse Serum for 30 minutes at RT.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with diluted Vector ABC Elite System for 30    minutes at RT.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with Pierce Metal Enhanced DAB for 5 minutes-   Rinse in Running Tap water for 5 minutes.-   Counterstain with Mayers Hematoxylin for 1 minute.-   Rinse in Running Tap water for 5 minutes.-   Blue Hematoxylin with Richard-Allan Bluing Reagent for 1 minute.-   Rinse in Running Tap water for 2 minutes.-   Dehydrate, clear and mount in synthetic mounting media.    Immunohistochemistry Procedure: Sialyl Lewis X

Antibody: Mouse anti-Sialyl Lewis X

Clone: KM93

Supplier: Chemicon

Catalog No. MAB2096

Ig Species: Mouse

IHC Method: Paraffin

Pretreatment: DAKO Target Retrieval

IHC Handling: Autostainer

Isotype: Mouse IgM

Procedure Species Human

IgG Concentration: 100 ug/ml

Normal Procedure:

-   Deparaffinize and hydrate to distilled water.-   Quench endogenous peroxidase activity with KPL Blocking    Solution—dilute concentrate 1:10 in dH2O, RT for 4 minutes.-   Rinse in Distilled water for 5 minutes.-   Incubate in DAKO Target Retrieval (S1700) preheated to 99 degrees    for 20 minutes in a boiling water bath. Remove from boiling bath and    let cool for 20 minutes.-   Block endogenous biotin with Vector Avidin Biotin Blocking System.-   Block with 10% Normal Horse Serum for 30 minutes at RT.-   Incubate sections with Mouse Monoclonal Sialyl Lewis X antibody    diluted to 5 ug/ml with 10% normal Horse Serum for 60 minutes at RT.    -   Use a mouse isotype IgM diluted at 5 ug/ml in 10% Normal Horse        Serum for the negative control.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with Vector biotinylated horse anti-mouse    antibody; 1:200 diluted in 10% Normal Horse Serum for 30 minutes at    RT.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with diluted Vector ABC Elite System for 30    minutes at RT.-   Rinse with TBS: 2 changes, 5 minutes each.-   Incubate sections with Pierce Metal Enhanced DAB for 5 minutes-   Rinse in Running Tap water for 5 minutes.-   Counterstain with Mayers Hematoxylin for 1 minute.-   Rinse in Running Tap water for 5 minutes.-   Blue Hematoxylin with Richard-Allan Bluing Reagent for 1 minute.-   Rinse in Running Tap water for 2 minutes.-   Dehydrate, clear and mount in synthetic mounting media.    Experimental Results:

Experiments were conducted using the methods and materials describedabove. Results of these experiments are illustrated in FIGS. 6-13, asdiscussed below.

FIG. 6 provides a summary chart of the data obtained in analyzing 28colon or colorectal cancer cell lines for sensitivity or resistance toapoptotic activity of Apo2L (+0.5% fetal bovine serum “FBS” or 10% FBS)or DR5 monoclonal antibody “mab”, cross-linked “XL” or not crosslinked,+0.5% fetal bovine serum “FBS” or 10% FBS) and expression of FUT 3, FUT6, Sialyl lewis A and Sialyl lewis X.

FIG. 7 provides a comparison of sensitivity of various colon orcolorectal cancer cell lines to DR5 antibody and the expression of FUT3, as measured by quantitative PCR).

FIG. 8 provides a comparison of various colon or colorectal cancer celllines for sensitivity or resistance to DR5 antibody (plus cross-linker)and expression of sialyl lewis X or A, as determined by FACS.

FIG. 9A shows a Spearman Rank Correlation test analyzing sensitivity orresistance of various colon or colorectal cancer cell lines andcorrelation to expression of FUT3.

FIG. 9B shows the results of a Fisher's Exact test analyzing sensitivity(“sens”) or resistance (“res”) of the various colon or colorectal cancercell lines and the statistical significance between FUT 3 and sialyllewis A/X expression and sensitivity of the respective cell lines to DR5antibody apoptotic activity.

FIG. 10 provides a comparison of various colon or colorectal cancer celllines for expression of DcR1 or DcR2 receptors (as determined byquantitative PCR) and the status (sensitive or resistant) of certaincell lines to Apo2L or DR5 antibody.

FIG. 11 provides a comparison of various colon or colorectal cancer celllines for expression of DcR1 or DcR2 receptors (as determined by FACS)and the status (sensitive or resistant) of certain cell lines to Apo2Lor DR5 antibody.

FIG. 12 shows immunohistochemical staining for sialyl lewis A and X onfour colorectal cancer cell lines, CaCo 2 (Colo2), SW 1417, DLD-1, andColo 205, and its correlation to expression of sialyl Lewis A and X asmeasured by FACS and its correlation to sensitivity to Apo2L/TRAIL.Colorectal cancer cell lines Colo 2 and SW1417 show no and weakstaining, respectively, or sialyl Lewis antigens, are negative andweakly positive, respectively, by FACS and are resistant to Apo2L/TRAIL.Colorectal cancer cells lines DLD-1 and Colo 205 show moderate andstrong staining, respectively for sialyl Lewis antigens, are moderatelyand strongly positive, respectively, by FACS and are sensitive toApo2L/TRAIL.

FIG. 13 shows a summary of IHC experiments demonstrating expression ofsialyl Lewis A and X in tissue samples of normal colon mucosa, normalliver tissue, primary colon cancer, and colon cancer metastases. Tissuesamples of normal colon and primary colon cancer arrayed in a tissuemicroarray were tested in the IHC experiment, while tissue samples ofthe normal liver and metastatic colon cancer were on individual glassslides. The prevalence of expression of sialyl Lewis A and X and theimmunohistochemical staining intensity increases from normal colontissue to primary colon cancer to metastatic colon cancer. The normalliver cells did not stain for either sialyl Lewis A or X.

What is claimed is:
 1. A method of treating cancer in a mammal,comprising the steps of: obtaining a cancer tissue or cancer cell samplefrom said mammal; examining the tissue or cell sample to detectexpression of one or more biomarkers selected from the group offucosyltransferase 3, fucosyltransferase 6, sialyl Lewis A and/or Xantigen(s), and subsequent to detecting expression of said one or morebiomarkers, administering to said mammal an effective amount ofApo2L/TRAIL.
 2. The method of claim 1 wherein said expression of one ormore biomarkers is examined by detecting mRNA expression offucosyltransferase 3 or fucosyltransferase
 6. 3. The method of claim 1wherein said expression of one or more biomarkers is examined byimmunohistochemistry to detect expression of sialyl Lewis A and/or Xantigen(s).
 4. The method of claim 1 further comprising the step ofexamining expression of DR4, DR5, DcR1 or DcR2 receptors in said tissueor cell.
 5. The method of claim 1 wherein said cancer cells or tissuecomprises colon, colorectal, gastrointestinal, or pancreatic cancercells or tissue.
 6. The method of claim 1 wherein an effective amount ofApo2L/TRAIL polypeptide comprising amino acids 114-281 of FIG. 1 (SEQ IDNO:1) is administered to said mammal.
 7. The method of claim 1 wherein achemotherapeutic agent(s) or radiation therapy is also administered tosaid mammal.
 8. The method of claim 1 wherein a cytokine, cytotoxicagent or growth inhibitory agent is also administered to said mammal. 9.The method of claim 1 wherein said Apo2L/TRAIL polypeptide is linked toa polyethylene glycol molecule.
 10. The method of claim 6 wherein saidApo2L/TRAIL polypeptide consists of amino acids 114-281 of FIG. 1 (SEQID NO:1).
 11. A method of treating colon or colorectal cancer in amammal, comprising the steps of: obtaining a colon or colorectal cancercell sample from said mammal; examining the cancer cell sample to detectexpression of one or more biomarkers selected from the group offucosyltransferase 3, fucosyltransferase6, sialyl Lewis A and/or Xantigen(s), and subsequent to detecting expression of said one or morebiomarkers, administering to said mammal an effective amount ofApo2L/TRAIL.
 12. The method of claim 11 wherein said Apo2L/TRAILpolypeptide comprises amino acids 41-281 of FIG. 1 (SEQ ID NO:1) or afragment thereof which has apoptotic activity.
 13. The method of claim12 wherein said Apo2L/TRAIL polypeptide is linked to a polyethyleneglycol molecule.
 14. The method of claim 12 wherein said Apo2L/TRAILpolypeptide comprises amino acids 114-281 of FIG. 1 (SEQ ID NO:1).